Developmental analysis of teosinte glume architecture1: A key locus in the evolution of maize (Poaceae)

A key event in the evolution of maize from teosinte was a reduction in the cupulate fruitcase and softening of the glumes, which increased the accessibility of kernels for harvest. The teosinte glume architecture1 (tga1) locus largely controls this difference between maize and teosinte, and thus may have played a pivotal role in maize evolution. The teosinte allele (tga1+teosinte) lengthens inflorescence internodes, shortens rachillae, and makes glumes longer, thicker, and harder. Developmental characterization of morphometric traits reveals that differences among genotypes are apparent early in female inflorescence development. Increased hardening in glumes homozygous for tga1+teosinte is correlated with a thicker abaxial mesoderm of lignified cells. Silica deposition in the abaxial epidermal cells of the glumes is also affected. In the maize background, glumes homozygous for tga1+teosinte deposit silica in both the short and long cells of the glume epidermis, whereas glumes homozygous for the maize allele (Tga1+Maize) concentrate silica only in the short cells. Silica deposition also appears to be affected by genetic background. The effects of tga1 appear largely to explain the differences in glume induration between maize and teosinte. The diverse pleiotropic effects of tga1 suggest that it is regulatory in nature.     

Effect of environment on the early steps of ear initiation in maize (Zea mays L.)

The effects of environmental conditions on ear-shoot initiation have been investigated in three inbred genotypes of Zea mays L. which are used for seed production. Scanning electron microscopy (SEM) and binocular examination during the vegetative phase showed that axillary meristems are initiated at the same rate as the leaf primordia on the apical meristem, but with a delay of 5.6–7.0 plastochrons, depending on the genotype. Furthermore, the topmost axillary meristem is initiated on the same day as the tassel, whatever the genotype. One of the inbreds (B22) used in this study has been reported to exhibit, in field conditions, a reproductive failure affecting car initiation, causing the topmost car to be replaced at maturity by a sterile, leaf-like, structure. Scanning electron microscopic study of the formation of the abnormal axillary buds indicated that ear failure resulted from the early collapse of the axillary meristem followed by elongation of the prophyll or of the meristem itself. Using controlled environments, ear abortion was mimicked by a chilling treatment (10°C), given just before tassel initiation. Other factors, such as high irradiance and flooding, enhanced the abortive response. The critical stage for the main car was just before the initiation of the topmost axillary meristem which also corresponded to tassel initiation. Chilling the plants before or after tassel initiation either induced an acclimation response or had no effect. The three inbreds showed differential responses to the stress treatment, indicating that a genetic factor is implicated as well. It is suggested that chilling causes a perturbation of apical dominance which, in the responsive genotypes, represses axillary meristem development. The use of a stress-sensitive inbred such as B22 as a model system could yield some interesting clues to the mechanism of endogenous control of ear initiation in maize.     

Early inflorescence and floral development in Zea mays land race Chapalote (Poaceae)

Tassel and ear primordia were collected from greenhouse-grown specimens of the Mexican maize landrace Chapalote and prepared for scanning electron microscopic (SEM) examination. Measurements of inflorescence apices and spikelet pair primordia (spp) were made from SEM micrographs. Correlation of inflorescence apex diameter with number of spikelet ranks showed no significant difference between tassels and ears, except at the two-rank level where the ear apical meristem had a significantly smaller diameter than corresponding two-ranked tassels. Within individual inflorescences, spp in different ranks enlarged at comparable rates, although the rates from one ear to the next along the stem differed. In both tassels and ears, spp divide to form paired sessile and pedicellate spikelet primordia when the spp is 150 μm wide; ear axes are significantly thicker than tassel axes at the time of bifurcation. The similarities in growth between ear and tassel primordia lend further support to the hypothesis that both the maize tassel and ear are derived from a common inflorescence pattern, a pattern shared with teosinte. Inflorescence primordial growth also suggests that a key character difference between teosinte and maize, distichous vs. polystichous arrangement of spikelets, may be related to size of the apical dome and/or rate of primordium production by the apical meristem. There appears to be more than a single morphological event in the shift from vegetative to reproductive growth. The evocation of axillary buds (ears) is independent of, and temporally separated from, the transition to flowering at the primary shoot apex (tassel).     

Localization of the genes controlling B chromosome transmission rate in maize (Zea mays ssp. mays, Poaceae)

In previous papers we found that the frequency of B chromosomes in native races of maize varies considerably in different populations. Moreover, we found genotypes that control high and low transmission rates (TR) of B chromosomes in the Pisingallo race. In the present work crosses were made to determine whether the genes controlling B-TR are located on the normal chromosome set (As) or on the B chromosomes (Bs). We made female f.0B × male m.2B crosses between and within high (H) and low (L) B-TR groups. The Bs were transmitted on the male side in all cases. The mean B-TR from the progeny of f.0B (H) × m.2B (H) and f.0B (H) × m.2B (L) crosses was significantly higher than that from f.0B (L) × m.2B (L) and f.0B (L) × m.2B (H) crosses. The results show that the B-TR of the crosses corresponds to the H or L B-TR of the 0B female parents irrespective of the Bs of the male parent. This indicates that B-TR is genetically controlled by the 0B female parent and that these genes are located on the A chromosomes.     

Lax midrib1-O, a systemic, heterochronic mutant of maize

Lxm1-O, a dominant EMS (ethyl methanesulfonate) induced mutation in maize (Zea mays, Poaceae), was originally reported to affect the blade/sheath boundary over the midrib region of the leaf. Here we present a more extensive analysis of the Lxm phenotype in nine different inbred lines. Lxm leaves are longer and narrower, and can initiate ectopic leaves. Additionally, Lxm1-O affects all plant organs observed. Compared to wild-type siblings, Lxm plants have fewer nodes, basal displacement of reproductive structures, and advance more quickly to the reproductive phase. We address questions as to whether Lxm1-O abbreviates a specific developmental phase, using hair, wax, and ear node data. We found that each phase was affected, although to varying degrees, depending on the inbred line. We interpret Lxm1-O to be a heterochronic mutation, causing the developmental acceleration of each phase of the shoot. Lxm1-O is novel, since other systemic heterochronic maize mutants prolong the juvenile phase, thereby extending shoot development. We discuss the importance of heterochronic mutations in the context of morphological evolution.     

Genome size and numerical polymorphism for the B chromosome in races of maize (Zea mays ssp. mays, Poaceae)

Twenty-one native populations (1120 individuals) of maize from Northern Argentina were studied. These populations, which belong to 13 native races, were cultivated at different altitudes (80-3620 m). Nineteen of the populations analyzed showed B chromosome (Bs) numerical polymorphism. The frequency of individuals with Bs varied from 0 to 94%. The number of Bs per plant varied from 0 to 8 Bs, with the predominant doses being 0, 1, 2, and 3. Those populations with varying number of Bs showed a positive and statistically significant correlation of mean number of Bs with altitude. The DNA content, in plants without Bs (A-DNA)(2n = 20), of 17 populations of the 21 studied was determined. A 36% variation (5.0-6.8 pg) in A-DNA content was found. A significant negative correlation between A-DNA content and altitude of cultivation and between A-DNA content and mean number of Bs was found. This indicates that there is a close interrelationship between the DNA content of A chromosomes and doses of Bs. These results suggest that there is a maximum limit to the mass of nuclear DNA so that Bs are tolerated as long as this maximum limit is not exceeded.     

Class II tassel seed mutations provide evidence for multiple types of inflorescence meristems in maize (Poaceae)

The tassel seed mutations ts4 and Ts6 of maize cause irregular branching in its inflorescences, tassels, and ears, in addition to feminization of the tassel due to the failure to abort pistils. A comparison of the development of mutant and wild-type tassels and ears using scanning electron microscopy reveals that at least four reproductive meristem types can be identified in maize: the inflorescence meristem, the spikelet pair meristem, the spikelet meristem, and the floret meristem. ts4 and Ts6 mutations affect the fate of specific reproductive meristems in both tassels and ears. ts4 mutants fail to form spikelet meristems from spikelet pair meristems. Ts6 mutants are delayed in the conversion of certain spikelet meristems into floret meristems. Once floret meristems are established in both of these mutants, they form florets that appear normal but fail to undergo pistil abortion in the tassel. The abnormal branching associated with each mutant is suppressed at the base of ears, permitting the formation of normal, fertile spikelets. The classification of the different types of reproductive meristems will be useful in interpretation of gene expression patterns in maize. It also provides a framework for understanding meristem functions that can be varied to diversify inflorescence architectures in the Gramineae.     

Biomechanical analysis of the Rolled (RLD) leaf phenotype of maize

The pleiotropic effects of the Rld1-O/+ mutation of Zea mays (Poaceae) on leaf phenotype include a suppression of normal transverse unrolling, a reversed top/bottom epidermal polarity, and an apparently straighter longitudinal shape. According to engineering shell theory, there might be mechanical coupling between transverse and longitudinal habit, i.e., the leaf rolling itself might produce the longitudinal straightening. We tested this possibility with quantitative curvature measurements and mechanical uncoupling experiments. The contributions of elastic bending under self weight, mechanical coupling, and rest state of leaf parts to the longitudinal and transverse habit were assessed in Rld1-O/+ mutants and a population of sibling +/+ segregants. Elastic bending and curvature coupling are shown to be relatively unimportant. The Rld1-O/+ mutation is shown to alter not only the unrolling process, but also the developmental longitudinal curving in the growing leaf, leading to a straighter midrib and a rolled lamina. The Rld1-O/+ mutant is thus a suitable model to study the relation between tissue polarity and differential curvature development in the maize leaf. Since on the abaxial side of the leaf, more abundant sclerenchyma is found in +/+ than in Rld1-O/+, a gradient in sclerification may contribute to the development of midrib curvature.     

Fertilization in maize indeterminate gametophyte1 mutant

Summary. Mature embryo sacs of the maize mutant indeterminate gametophyte1 displayed different cellular patterns compared to those of the wild type. About 40% of the ig1 embryo sacs contained three or more synergids and two or more egg cells at the micropylar end. During fertilization in embryo sacs with two synergids, both of them frequently degenerated and were penetrated by two pollen tubes. 75% of the embryo sacs containing three or more synergid cells were penetrated by two or more pollen tubes, although most of them had only one degenerated synergid. Multiple fusions between the sperm cells and eggs frequently occurred in the same embryo sac, which subsequently generated multiple embryos. There were two or more central cells in about 33% of ig1 embryo sacs. The largest central cell was usually adjacent to the egg apparatus and contained two unfused polar nuclei, while those extra central cells located at the chalazal end usually had a single nucleus. Fertilization occurred only between the male gamete and the largest binucleate central cell. The extra central cells eventually degenerated after fertilization.Present address: GI Basic Research Center, Mayo Clinic, Rochester, Minnesota, U.S.A.Correspondence and reprints: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, Peoples Republic of China.     

Toward Molecular Understanding of In Vitro and In Planta Shoot Organogenesis

Shoot organogenesis, one of the in vitro plant regeneration processes that occur during in vitro micropropagation, is used in the study of plant development. Morphological, physiological, and molecular aspects of in vitro shoot organogenesis have been extensively studied for over 50 years. Because of the research progress in plant genetics and molecular biology, our understanding of in planta and in vitro shoot meristem development, the cell cycle and cytokinin signal transduction has advanced significantly. These research advances provide useful information as well as molecular tools to study further the genetic and molecular aspects of shoot organogenesis. A number of key molecular markers, genes, and pathways have been shown to play a critical role in the process of in vitro shoot organogenesis. Furthermore, these studies reveal that in vitro shoot organogenesis, as with in planta shoot development, is a complex, well-coordinated developmental process, given that the induction of a single molecular event is likely to be insufficient to induce the entire process. Continued study is required to identify additional molecular events that trigger dedifferentiation and act as developmental switches for de novo shoot development.     

Genetics of resistance to the pink stem borer (Sesamia nonagrioides) in maize (Zea mays)

Our knowledge of the genetics of resistance to the pink stem borer ( Sesamia nonagrioides ) in maize ( Zea mays ) is restricted to a few crosses among maize inbreds. The objectives of this study were to enlarge our understanding of the genetics of traits related to damage by pink stem borer and yield under infestation and to use generation means analyses to compare per se and testcross performance for detecting epistatic effects. All generations, either per se or crossed to testers, were evaluated in a 10 × 10 triple lattice design under artificial infestation with S. nonagrioides in 2005 and 2006. Most traits fit an additive–dominance model; but evidence for epistasis for resistance and yield under infestation was shown. Epistasis, in general, did not appear to play an important role in the inheritance of yield under pink stem borer infestation. However, the epistasis contribution to maize yield performance could be important in some outstanding crosses such as EP42 × A637. Testcross generation means revealed epistatic effects undetected by the generation means analysis, but neither method was able to eliminate dominance effects that could prevail over epistatic effects.     

Maturation of stamens and ovaries on cultured ear inflorescences of maize (Zea mays L.)

Observations were made on the maturation of stamens and ovaries from cultured maize (Zea mays L.) ear inflorescences. Immature ears (5.1–10.0 mm long) of maize were cultured in kinetin medium to study microsporogenesis and pollen maturation in developing stamens. Male spikelets developed on ears cultured in kinetin medium. Meiosis-I began by 7 days of culture in the developing anthers and the mature tri-nucleate pollen grains were developed by 20 days of culture. Further, kinetin was required in the culture medium for at least initial 5 days to obtain the microspores in differentiated stamens.To observe the embryosac formation in developed ovaries, ears were cultured in control, kinetin (10.1–15.0 mm long ears) medium, and kinetin + gibberellic acid (5.1–10.0 mm long ears) medium. Formation of embryosacs was noticed in the developed ovaries which were sampled after 20 days of culture. This differential flower development using two growth regulators provides an opportunity to uncover the biochemistry and physiology of micro- and mega-gametophyte development in maize.     

Tissue distribution and developmental patterns of NADH-dependent and ferredoxin-dependent glutamate synthase activities in maize (Zea mays) kernels

Both NADH-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14) and ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) activities were present in the endosperm, embryo, pedicel and pericarp of maize ( Zea mays L. var. W64A × A619) kernels. The endosperm contained the highest proportions of each activity on a per tissue basis. In the endosperm, NADH-GOGAT and Fd-GOGAT activities increased 12- and 2.5-fold, respectively, during early zein accumulation. NADH-GOGAT and Fd-GOGAT activities were expressed in the upper, middle and lower portions of the endosperm in a manner that paralleled but preceded zein accumulation. Maize endosperm NADH-GOGAT was purified 159-fold using ammonium sulfate fractionation, anion exchange chromatography and dye-ligand chromatography. Apparent K_m values for glutamine, α-ketoglutarate and NADH were 850, 19 and 1 μM, respectively. The results are consistent with endosperm GOGAT functioning to redistribute nitrogen from glutamine, the predominant nitrogenous compound delivered to the endosperm, into other amino acids needed for storage protein synthesis.     

Genetic control of B chromosome transmission rate in Zea mays ssp. mays (Poaceae)

We selected genotypes of high and low B chromosome transmission rate (TR) in a native race of maize (Pisingallo) from northwest Argentina. We made 20 female OB male IB and 20 f.IB m.OB crosses. The former (GOm) showed a large variation of B TR, with a mean of TR ± SE = 0.52 ± 0.06, ranging from 0.17 to 0.98. In the latter (GOf) the mean was TR = 0.47 ± 0.02 ranging from 0.31 to 0.58. Plants showing the highest and the lowest TR were selected to constitute the progenitors of the G1 generations. We made 19 f.0B m.2B crosses, studying 24–30 plants per cross. The TR of the high (H) and low (L) lines in Glm (GlmH and GlmL) significantly differed (TRH = 0.65 ± 0.03, TRL = 0.40 ± 0.01), indicating that the H and L lines are different groups. The large variation in male TR suggests that preferential fertilization of gametes carrying B chromosomes does not always occur. We also selected plants showing high and low TR in the progenies of f.lB m.OB crosses (GOf), and made 24 f.1B m.0B crosses, studying 23–30 plants per cross. The TRs of the H and L lines in G1f (GlfH and GlfL) were significantly different (TRH = 0.48 ± 0.025, TRL = 0.40 ± 0.02). The TRs in G0f and G1fL were significantly different (TR = 0.47 ± 0.02 and 0.40 ± 0.02, respectively), while this was not the case between G0f and G1fH. Our results demonstrate the existence of genotypes controlling B TR in this native population of maize.     

Genome assembly of the Chinese maize elite inbred line RP125 and its EMS mutant collection provide new resources for maize genetics research and crop improvement

Maize is an important crop worldwide, as well as a valuable model with vast genetic diversity. Accurate genome and annotation information for a wide range of inbred lines would provide valuable resources for crop improvement and pan-genome characterization. In this study, we generated a high-quality de novo genome assembly (contig N50 of 15.43 Mb) of the Chinese elite inbred line RP125 using Nanopore long-read sequencing and Hi-C scaffolding, which yield highly contiguous, chromosome-length scaffolds. Global comparison of the RP125 genome with those of B73, W22, and Mo17 revealed a large number of structural variations. To create new germplasm for maize research and crop improvement, we carried out an EMS mutagenesis screen on RP125. In total, we obtained 5818 independent M2 families, with 946 mutants showing heritable phenotypes. Taking advantage of the high-quality RP125 genome, we successfully cloned 10 mutants from the EMS library, including the novel kernel mutant qk1 (quekou: “missing a small part” in Chinese), which exhibited partial loss of endosperm and a starch accumulation defect. QK1 encodes a predicted metal tolerance protein, which is specifically required for Fe transport. Increased accumulation of Fe and reactive oxygen species as well as ferroptosis-like cell death were detected in qk1 endosperm. Our study provides the community with a high-quality genome sequence and a large collection of mutant germplasm.     

Properties of glutamate dehydrogenase from developing maize endosperm

Glutamate dehydrogenase (EC 1.4.1.3) activity was assayed in homogenates of maize ( Zea mays L. inbred lines Oh43 and Oh43o₂) endosperm during development. During the period 20–35 days after pollination anabolic (aminative) activities were higher than catabolic (deaminating) ones. In order to study the regulation of GDH activity, glutamine or glutamate were injected into the ear peduncle before sample harvesting. The amination and deamination reactions showed similar behaviour with different nitrogen sources: glutamine increased, whereas glutamate decreased, both aminative and deaminative reactions. Partially purified enzyme was active with NADH and NADPH in a ratio 9:1. In Tris-HCl buffer a broad optimum at pH 7.6–8.9 and pH 6.8–8.9 was observed with NADH and NADPH, respectively, NADH activity was activated by Ca²⁺. Saturation curves for (NH₄)₂SO₄ and NADH showed normal Michaelis-Menten kinetics in the presence of 1 m M Ca²⁺, but substrate inhibition occurred without Ca²⁺. The enzyme was inactivated by EDTA. The effect of EDTA was reversed by Ca²⁺ and Mn²⁺, but not by Cu₂₊ and Mg²⁺.     

Regulation of glutamate dehydrogenase activity by amino acids in maize seedlings

Root or secondary leaf segments from maize ( Zea mays L. cv. Ganga safed-2) seedlings were incubated with 9-amino acids and two amides separately, each at 5 m M for 24 h, to study their effects on glutamate dehydrogenase (GDH) activity. Most of the compounds tested inhibited the specific activity of NADH-GDH and increased that of NAD⁺-GDH in the roots in the presence as well as in the absence of ammonium. In the leaves, such effects were recorded only with a few amino acids. Total soluble protein in the root and leaf tissues increased with the supply of most of the amino compounds. The effect of glutamate on enzyme activity and protein was concentration dependent in both tissues. When the enzyme extracts from root or leaf tissues were incubated with some of the amino acids, NADH-GDH declined while NAD⁺-GDH increased in most cases. The inhibition of NADH-GDH increased with increasing concentration of cysteine from 1 to 5 m M . The experiments demonstrate that most of the amino acids regulated GDH activity, possibly through some physicochemical modulation of the enzyme molecule.