Gametophytic expression of genes controlling endosperm development in maize

Summary An indirect approach was adopted to select viable mutants affecting the male gametophytic generation in maize. This approach consists of a selection of endosperm defective mutants followed by a test for gametophytic gene expression, based on the distortion from mendelian segregation and on the measurement of pollen size and pollen sterility. The material used consisted of 34 endosperm defective viable mutants introgressed in B37 genetic background. Complementation tests indicated that the mutation in the collection of mutants affected different genes controlling endosperm development. The study of the segregation in F2 revealed four classes of de (defective endosperm) mutants: (1) mutants in which the mutation does not affect either gametophytic development or function; (2) mutants in which the effect on the gametophyte affects pollen development processes; (3) mutants showing effects on both pollen development and function, and (4) mutants where only pollen tube growth rate is affected. Positive and negative interactions between pollen and style were detected by means of mixed pollination (pollen produced by de/de plants and pollen from an inbred line used as a standard and carrying genes for colored aleurone), on de/de and de/ + plants. Positive interactions were interpreted as methabolic complementation between defective pollen and normal styles.     

Identification of genes alternatively spliced in developing maize endosperm

• The process of alternative splicing is critical for the regulation of growth and development of plants. Thus far, little is known about the role of alternative splicing in the regulation of maize (Zea mays L.) endosperm development.
• RNA sequencing (RNA‐seq) data of endosperms from two maize inbred lines, Mo17 and Ji419, at 15 and 25 days after pollination (DAP), respectively, were used to identify genes that were alternatively spliced during endosperm development. Intron retention (IR) in GRMZM2G005887 was further validated using PCR and re‐sequencing technologies.
• In total, 49,000 alternatively spliced events and ca. 20,000 alternatively spliced genes were identified in the two maize inbred lines. Of these, 30 genes involved in amino acid biosynthesis and starch biosynthesis were identified, with IR occurring only in a specific sample, and were significantly co‐expressed with ten well‐known genes related to maize endosperm development. Moreover, IR in GRMZM2G005887, which encodes a cysteine synthase, was confirmed to occur only in the endosperm of Mo17 at 15 DAP, resulting in the retention of a 121‐bp fragment in its 5′ untranslated region. Two cis‐acting regulatory elements, CAAT‐box and TATA‐box were observed in the retained fragment in Mo17 at 15 DAP; this could regulate the expression of this gene and influence endosperm development.
• The results suggest that the 30 genes with IR identified herein might be associated with maize endosperm development, and are likely to play important roles in the developing maize endosperm.

     

Increased hexokinase levels in endosperm of mutant maize

Hexokinase activity was measured in endosperms of shrunken-2 (sh₂) and starchy maize. Initial increases in hexokinase were observed for developing endosperms of both genotypes, and the enzyme declined in both as the seeds matured. A higher level of hexokinase was observed in developing sh₂ than in starchy endosperm. This difference persisted throughout maturation and occurred also in germinating seeds. Soluble hexokinase activity per endosperm continued to increase in sh₂ for about 8 days (22–30 days after pollination) after the enzyme in starchy endosperm had attained maximum activity and begun to decline. Hexokinase was predominantly soluble in both genotypes so the differences observed are not due to altered distribution of enzyme between particulate and soluble fractions.     

Tissue-specific expression in transgenic maize of four endosperm promoters from maize and rice

The tissue-specific, developmental, and genetic control of four endosperm-active genes was studied via expression of GUS reporter genes in transgenic maize plants. The transgenes included promoters from the maize granule-bound starch synthase (Waxy) gene (zmGBS), a maize 27 kDa zein gene (zmZ27), a rice small subunit ADP-glucose pyrophosphorylase gene (osAGP) and the rice glutelin 1 gene (osGT1). Most plants had a transgene expression profile similar to that of the endogenous gene: expression in the pollen and endosperm for the zmGBS transgene, and endosperm only for the others. Histological analysis indicated expression initiated at the periphery of the endosperm for zmGBS, zmZ27 and osGT1, while osAGP transgene activity tended to start in the lower portion of the seed. Transgene expression at the RNA level was proportional to GUS activity, and did not influence endogenous gene expression. Genetic analysis showed that there was a positive dosage response with most lines. Activity of the zmGBS transgene was threefold higher in a low starch (shrunken2) genetic background. This effect was not seen with zmZ27 or osGT1 transgenes. The expression of the transgenes is discussed relative to the known behaviour of the endogenous genes, and the developmental programme of the maize endosperm     

Free and bound indole-acetic acid is low in the endosperm of the maize mutantdefective endosperm-B18

Summary The maize mutant defective endosperm-B18 (de ^*-B18), which is recessive to its wildtype, accumulates substantially less dry matter in the endosperm than its normal counterpart. Both free and bound indole-acetic acid (IAA) content has been measured at 5 different developmental stages. In endosperm tissue, the level of IAA is at least 15 times lower in the mutantde ^* -B18 than in the wildtype. The situation found in the diploid tissues is somewhat different: in the mature embryo the level of total IAA is lower in the mutant than in the wildtype, while in 4-day old seedlings the level of total IAA is, to some degree, similar in both genotypes. Naphthalene-acetic acid (NAA), a stable synthetic auxin which mimics IAA in its biochemical effects, is able to normalize the seed weight of the mutant when applied to developing grains. The results favor the conclusion that in maize endosperm the mutationde ^* -B18 is involved in IAA metabolism.     

Evidence for the genetic control of lysine catabolism in maize endosperm

A split pollination was used to produce normal (Su su su O2 o2 o2) and high lysine double mutant sugary opaque-2 (su su su o2 o2 o2) endosperms on the same ear of sugary opaque-2 maize plants. Amino acids were determined in the vascular sap of the ear peduncle. Lysine content in the sap was compared with lysine stored in both normal and sugary opaque-2 endosperm during kernel filling. Lysine content in the ear peduncle sap could account for all lysine found in both endosperms. Preformed lysine is highly catabolized in the normal endosperm, but not in the high lysine sugary opaque-2 endosperm. The rate of lysine breakdown appears to be an important mechanism by which the high lysine mutant controls lysine level in maize endosperm.     

Transgenic rice lines expressing maize C1 and R-S regulatory genes produce various flavonoids in the endosperm

Flavonoids, compounds that possess diverse health-promoting benefits, are lacking in the endosperm of rice. Therefore, to develop transgenic lines that produce flavonoids, we transformed a white rice cultivar, Oryza sativa japonica cv. Hwa-Young, with maize C1 and R-S regulatory genes. Expression of these transgenes was restricted to the endosperm using the promoter of a rice prolamin gene. The pericarp of the C1/R-S homozygous lines became dark brown in accordance with their maternal genotype, whereas the endosperm turned chalky, similar to the opaque kernel phenotype. Analysis via high-performance liquid chromatography (HPLC) revealed that numerous kinds of flavonoids were produced in these transgenic kernels. To identify individual flavonoids, the number of HPLC peaks was reduced through moderate acid hydrolysis, followed by ethyl acetate partitioning. Amongst the major flavonoids, dihydroquercetin (taxifolin), dihydroisorhamnetin (3'-O-methyl taxifolin) and 3'-O-methyl quercetin were identified through liquid chromatography/mass spectrometry/mass spectrometry and nuclear magnetic resonance analyses. Fluorescence labelling with diphenylboric acid showed that the flavonoids were highly concentrated in the cells of four to five outer endosperm layers. More importantly, a high fluorescence signal was present in the cytosol of the inner endosperm layers. However, the overall signal in the inner layers was significantly lower because starch granules and protein bodies occupied most of the cytosolic space. Our estimate of the total flavonoid content in the transgenic kernels suggests that C1/R-S rice has the potential to be developed further as a novel variety that can produce various flavonoids in its endosperm.     

Protein breakdown and protease properties of germinating maize endosperm

Protein breakdown during germination of maize at 28° is closely correlated with the appearance of protease activity. In the first 2 days of germination, a slight disaggregation of only G₃ glutelins into more simple elements (albumin-globulins) can be observed. Between 2 and 2.5 days, there is extensive breakdown of all protein fractions, the rate of which coincides with the rate of appearance of proteolytic activity. After 2.5 days these phenomena slow down and the bulk of the endosperm proteins disappears. Three acid proteases in endosperm extracts of germinated grain (P₁₁, P₂₁ and P₂₂) have been isolated by affinity chromatography and gel filtration, and partially characterized. P₁₁ (MW 40 000) which is present in the ungerminated grain, cannot hydrolyse prolamins and is insensitive to reducing agents. P₂₁ (MW 36 000) and P₂₂ (MW 12 000), which appear on day 3 of germination, can degrade prolamins in vitro. Reducing agents enhance their activity and prevent their aggregation or denaturation. Comparative assays with different substrates suggest our enzyme preparations are principally endotype proteases with little contaminating carboxypeptidase activity.     

Regulation of starch biosynthesis in normal and opaque-2 maize during endosperm development

The absolute activities of sucrose-UDP glucosyltransferase, glucose-6-phosphate ketoisomerase and soluble and bound ADPG-starch glucosyltransferase have been studied in normal and Opaque-2 maize endosperms during development. In general, the activities of these enzymes except sucrose-UDP glucosyltransferase were higher up to 20 days post-pollination and lower at the 30 day stage in Opaque-2 than in normal maize endosperms. However, sucrose-UDP glucosyltransferase activity was higher in normal maize endosperm up to the 20 day stage while it was lower at subsequent stages than in Opaque-2. It is suggested that the lower level of these enzymes, except sucrose-UDP glucosyltransferase, might be responsible for the reduced accumulation of starch in Opaque-2 endosperm during later stages of endosperm development.     

Evidence for factors regulating transfer cell-specific expression in maize endosperm

In maize, a layer of basal endosperm cells adjacent to the pedicel is modified for a function in solute transfer. Three genes specifically expressed in this region, termed the basal endosperm transfer layer (BETL-2 to -4), were isolated by differential hybridization. BETL-2 to -4 are coordinately expressed in early and mid-term endosperm development, but are absent at later stages. BETL-2 to -4 coding sequences all predict small (<100 amino acids), secreted, cysteine-rich polypeptides which lack close relatives in current database accessions. BETL-3 and BETL-1 display some sequence similarities with each other and to plant defensins. BETL-2 to -4 promoter regions were isolated and compared, revealing the presence of a promoter-proximal microsatellite repeat as the most highly conserved sequence element in each sequence. Electrophoretic mobility shift assays (EMSA) showed that specific BETL-2 to -4 promoter fragments competed for binding to the same DNA-binding activity in nuclear extracts prepared from maize endosperm. Although BETL-2 to -4 are only expressed in basal endosperm cells, the DNA-binding activities detected were of two types: distal endosperm-specific, or present in both basal and distal endosperm extracts. On the basis of these findings, a model to account for the coordinate regulation of BETL genes in endosperm cells is proposed.     

Structure and expression of zein genes of maize

Plant seed storage proteins are synthesized and deposited in endosperm or cotyledon tissue to serve an important physiological function at the onset of germination. Because of their abundance, they constitute an important factor for the amount and nutritional value of kernel proteins. The physiological, biochemical, and genetic properties of many storage proteins and their genes, in particular those of cereals and legumes, have been extensively studied in the past and the results have been summarized in several reviews.^1–6 More recently, representative genes coding for storage proteins have been isolated and are now being used in attempts to elucidate the mechanism of the regulated synthesis of storage proteins. The purpose of this review is to outline, using maize as an example, the recent progress made in this effort.     

The integration of mutant loci affecting maize endosperm development in a dense genetic map using an AFLP-based procedure

In this paper, 10 mutations conditioning the appearance of defective, miniature or collapsed endosperm, but with normal sporophyte development, were considered. Homozygous mutant kernels have reduced grain weight, kernel size, density and, in some of these, higher than normal seed protein content. The mutant loci were integrated into a high-resolution genetic map in order to associate them to specific genes. We have placed 1167 AFLP markers on a consensus map using IBM2 as a backbone and reaching an average of 1 marker every 1.9 cM. We have identified AFLP markers linked to all individual mutant alleles. BSA was adopted to screen the largest possible number of primer combinations on homozygous F₃ mutant and wild type plants. The ten mutant loci are linked to the closest AFLP or SSR markers with distances ranging from 0 to 17.9 cM. The genes we have defined by the existence of mendelian mutants can now be considered good candidates for testing the association to QT loci. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of programmed cell death in maize endosperm by abscisic acid

Cereal endosperm undergoes programmed cell death (PCD) during its development, a process that is controlled, in part, by ethylene. Whether other hormones influence endosperm PCD has not been investigated. Abscisic acid (ABA) plays an essential role during late seed development that enables an embryo to survive desiccation. To examine whether ABA is also involved in regulating the onset of PCD during endosperm development, we have used genetic and biochemical means to disrupt ABA biosynthesis or perception during maize kernel development. The onset and progression of cell death, as determined by viability staining and the appearance of internucleosomal DNA fragmentation, was accelerated in developing endosperm of ABA-insensitive vp1 and ABA-deficient vp9 mutants. Ethylene was synthesized in vp1 and vp9 mutant kernels at levels that were 2–4-fold higher than in wild-type kernels. Moreover, the increase and timing of ethylene production correlated with the premature onset and accelerated progression of internucleosomal fragmentation in these mutants. Treatment of developing wild-type endosperm with fluridone, an inhibitor of ABA biosynthesis, recapitulated the increase in ethylene production and accelerated execution of the PCD program that was observed in the ABA mutant kernels. These data suggest that a balance between ABA and ethylene establishes the appropriate onset and progression of programmed cell death during maize endosperm development.     

Isolation and sugar uptake characteristics of protoplasts from maize endosperm suspension cultures

The isolation and sugar uptake characteristics of protoplasts from maize ( Zea mays L.) endosperm-derived suspension cultures are described. In contrast with protoplasts from intact developing endosperm, which by virtue of their large size and high starch content are too fragile for sugar uptake experiments, suspension cultures yielded protoplasts capable of withstanding the necessary handling and centrifugations. Intactness of the protoplasts was demonstrated by dye exclusion or accumulation and latency of malate dehydrogenase activity. Uptake of radioactivity from [³H]-inulin did not increase with time, but that from [¹⁴C]-sugars increased over a wide range of external concentrations. Kinetics of fructose, glucose and sucrose uptake were biphasic, and the saturable components of uptake were eliminated by p -chloromercuribenzene sulfonate (PCMBS). Rates of uptake of sucrose and 1'-fluorosucrose were similar, confirming that hydrolysis by cell wall invertase contributes to sucrose uptake by the suspension cultures. The isolation of protoplasts from this tissue source will enable experimental access to plasma membrane sugar carriers which may exist in the intact maize endosperm.