Cyclin-dependent kinase complexes in developing maize endosperm: evidence for differential expression and functional specialization

Endosperm development in maize (Zea mays L.) and related cereals comprises a cell proliferation stage followed by a period of rapid growth coupled to endoreduplication. Regulation of the cell cycle in developing endosperm is poorly understood. We have characterized various subunits of cyclin-dependent kinase (CDK) complexes, master cell cycle regulators in all eukaryotes. A-, B-, and D-type cyclins as well as A- and B-type cyclin-dependent kinases were characterized with respect to their RNA and protein expression profiles. Two main patterns were identified: one showing expression throughout endosperm development, and another characterized by a sharp down-regulation with the onset of endoreduplication. Cyclin CYCB1;3 and CYCD2;1 proteins were distributed in the cytoplasm and nucleus of cells throughout the endosperm, while cyclin CYCD5 protein was localized in the cytoplasm of peripheral cells. CDKB1;1 expression was strongly associated with cell proliferation. Expression and cyclin-binding patterns suggested that CDKA;1 and CDKA;3 are at least partially redundant. The kinase activity associated with the cyclin CYCA1 was highest during the mitotic stage of development, while that associated with CYCB1;3, CYCD2;1 and CYCD5 peaked at the mitosis-to-endoreduplication transition. A-, B- and D-type cyclins were more resistant to proteasome-dependent degradation in endoreduplicating than in mitotic endosperm extracts. These results indicated that endosperm development is characterized by differential expression and activity of specific cyclins and CDKs, and suggested that endoreduplication is associated with reduced cyclin proteolysis via the ubiquitin–proteasome pathway.     

Water deficit in developing endosperm of maize: cell division and nuclear DMA endoreduplication

Water deficit severely decreases maize (Zea mays L.) kernel growth; the effect is most pronounced in apical regions of ears. The capacity for accumulation of storage material in endosperms is thought to he partially determined by the extent of cell division and endoreduplication (post-mitotic nuclear DNA synthesis). To gain a better understanding of the regulatory mechanisms involved, we have examined the effect of water deficit on cellular development during the post-fertilization period. Greenhouse-grown maize was subjected to water-limited treatments during rapid cell division [from 1 to 10days after pollination (DAP)] or rapid endoreduplication (9 to 15 DAP). The number of nuclei and the nuclear DNA content were determined with flow cytometry. Water deficit from 1 to 10 DAP substantially decreased the rate of endosperm cell division in apical-region kernels, but had little effect on middle-region endosperms. Rewatcring did not allow cell division to recover in apical-region endosperms. Water deficit from 9 to 15 DAP also decreased cell division in apical-region endosperms. Endoreduplication was not affected by the late treatment in either region of the car, but was inhibited by the early treatment in the apical region. In particular, the proportion of nuclei entering higher DN A-content size classes was reduced. We conclude that cell division is highly responsive to water deficit, whereas endoreduplication is less so. We also conclude that the reduced proportion of nuclei entering higher DNA-content size classes during endoreduplication is indicative of multiple control points in the mitotic and endoreduplication cycles.     

Fructan Accumulation and Sucrose Metabolism in Transgenic Maize Endosperm Expressing a Bacillus amyloliquefaciens SacB Gene

Over 40,000 species of plants accumulate fructan, [beta]-2-1- and [beta]-2-6-linked polymers of fructose as a storage reserve. Due to their high fructose content, several commercial applications for fructans have been proposed. However, plants that accumulate these polymers are not agronomically suited for large-scale cultivation or processing. This study describes the transformation of a Bacillus amyloliquefaciens SacB gene into maize (Zea mays L.) callus by particle bombardment. Tissue-specific expression and targeting of the SacB protein to endosperm vacuoles resulted in stable accumulation of high-molecular-weight fructan in mature seeds. Accumulation of fructan in the vacuole had no detectable effect on kernel development or germination. Fructan levels were found to be approximately 9-fold higher in sh2 mutants compared to wild-type maize kernels. In contrast to vacuole-targeted expression, starch synthesis and endosperm development in mature seeds containing a cytosolically expressed SacB gene were severely affected. The data demonstrate that hexose resulting from cytosolic SacB activity was not utilized for starch synthesis. Transgenic seeds containing a chimeric SacB gene provide further evidence that the dominant pathway for starch synthesis in maize endosperm is through uridine diphosphoglucose catalyzed by the enzyme sucrose synthase.     

Investigating the hows and whys of DNA endoreduplication

Endoreduplication is a form of nuclear polyploidization thatresults in multiple, uniform copies of chromosomes. This processis common in plants and animals, especially in tissues withhigh metabolic activity, and it generally occurs in cells thatare terminally differentiated. In plants, endoreduplicationis well documented in the endosperm and cotyledons of developingseeds, but it also occurs in many tissues throughout the plant.It is thought that endoreduplication provides a mechanism toincrease the level of gene expression, but the function of thisprocess has not been thoroughly investigated. Numerous observationshave been made of endoreduplication, or at least extra cyclesof S-phase, as a consequence of mutations in genes controllingseveral aspects of cell cycle regulation. However, until recentlythere were few studies directed at the molecular mechanismsresponsible for this specialized cell cycle. It is suggestedthat endoreduplication requires nothing more elaborate thana loss of M-phase cyclin-dependent kinase activity and oscillationsin the activity of S-phase cyclin-dependent kinase. Key words: Endoreduplication, gene expression, cell cycle regulation, cyclin-dependent kinase.     

A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality

Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.     

Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm

In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds.     

Cyclin-dependent kinase inhibitors in maize endosperm and their potential role in endoreduplication

Two maize (Zea mays) cyclin-dependent kinase (CDK) inhibitors, Zeama;KRP;1 and Zeama;KRP;2, were characterized and shown to be expressed in developing endosperm. Similar to the CDK inhibitors in Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), the maize proteins contain a carboxy-terminal region related to the inhibitory domain of the mammalian Cip/Kip inhibitors. Zeama;KRP;1 is present in the endosperm between 7 and 21 d after pollination, a period that encompasses the onset of endoreduplication, while the Zeama;KRP;2 protein declines during this time. Nevertheless, Zeama;KRP;1 accounts for only part of the CDK inhibitory activity that peaks coincident with the endoreduplication phase of endosperm development. In vitro assays showed that Zeama;KRP;1 and Zeama;KRP;2 are able to inhibit endosperm Cdc2-related CKD activity that associates with p13(Suc1). They were also shown to specifically inhibit cyclin A1;3- and cyclin D5;1-associated CDK activities, but not cyclin B1;3/CDK. Overexpression of Zeama;KRP;1 in maize embryonic calli that ectopically expressed the wheat dwarf virus RepA protein, which counteracts retinoblastoma-related protein function, led to an additional round of DNA replication without nuclear division.     

Developmental analysis of maize endosperm proteome suggests a pivotal role for pyruvate orthophosphate dikinase

Although the morphological steps of maize (Zea mays) endosperm development are well described, very little is known concerning the coordinated accumulation of the numerous proteins involved. Here, we present a proteomic study of maize endosperm development. The accumulation pattern of 409 proteins at seven developmental stages was examined. Hierarchical clustering analysis allowed four main developmental profiles to be recognized. Comprehensive investigation of the functions associated with clusters resulted in a consistent picture of the developmental coordination of cellular processes. Early stages, devoted to cellularization, cell division, and cell wall deposition, corresponded to maximal expression of actin, tubulins, and cell organization proteins, of respiration metabolism (glycolysis and tricarboxylic acid cycle), and of protection against reactive oxygen species. An important protein turnover, which is likely associated with the switch from growth and differentiation to storage, was also suggested from the high amount of proteases. A relative increase of abundance of the glycolytic enzymes compared to tricarboxylic acid enzymes is consistent with the recent demonstration of anoxic conditions during starch accumulation in the endosperm. The specific late-stage accumulation of the pyruvate orthophosphate dikinase may suggest a critical role of this enzyme in the starch-protein balance through inorganic pyrophosphate-dependent restriction of ADP-glucose synthesis in addition to its usually reported influence on the alanine-aromatic amino acid synthesis balance.     

Identification of quantitative trait loci that affect endoreduplication in maize endosperm

Endoreduplication in maize endosperm precedes the onset of starch and storage protein synthesis, and it is generally thought to influence grain filling. We created four backcross populations by reciprocally crossing the F₁ progeny of a cross between Sg18 and Mo17 to the parental inbreds, which differ in endoreduplication by two parameters—mean ploidy and percentage of endoreduplicated nuclei. This four-backcross design allowed us to estimate and test the additive and dominant genetic effects of quantitative trait loci (QTLs) affecting endoreduplication. An analysis of endosperm from the four backcross populations at 16 days after pollination using a modified triploid mapping approach identified three endosperm QTLs influencing mean ploidy and two endosperm QTLs affecting the percentage of endoreduplicated nuclei. Some of these QTLs may manifest their effects on endoreduplication via expression in the embryo. The QTLs detected display strong dominance or over-dominance and interacted epistatically with an embryo-expressed QTL. This helps to explain the genetic basis for transgressive segregation in the backcross progeny. Although the favorable alleles that increase mean ploidy and percentage of endoreduplicated nuclei can be contributed by both parents, the Mo17-derived alleles for endoreduplication were often dominant or over-dominant to the Sg18-derived allele. One QTL on chromosome 7 that may be expressed in both the embryo and endosperm exerted a pleiotropic effect on two different parameters of endoreduplication. The results from this study shed light on the regulation of endoreduplication in maize endosperm and provide a marker-assisted selection strategy for potentially improving grain yield. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. C. M. Coelho and S. Wu contributed equally to this work and should be considered as first authors.     

DNA endoreduplication in maize endosperm cells: the effect of exposure to short-term high temperature

DNA endoreduplication in Zea mays L. (cv. A619 × W64A) endosperm peaks between 16 and 18 d after pollination (DAP). The physiological function of DNA endoreduplication is not known but it is believed to be important in maize kernel development. In the present study, we investigated how 2, 4 or 6 d of high temperature (35 °C) affected DNA endoreduplication and maize kernel development in comparison with control kernels grown at 25 °C. Data were collected on fresh weight (FW), nuclei number, mitotic index, and DNA endoreduplication. Maize endosperm FW and nuclei number were reduced by exposure to 4 or 6 d of high temperature. At 18 DAP, the 2 d high temperature treatment (HTT) caused a reduction in FW and nuclei number, but had no effect on DNA endoreduplication and average DNA content per endosperm. However, when the exposure to high temperature was increased to 4 or 6 d, FW, nuclei number and the magnitude of DNA endoreduplication were progressively reduced, and the peak mitotic index was delayed compared with the control endosperm. At 18 DAP, the 4 d treatment showed 54·7% of the cells were 3 or 6 C, whereas only 41·2% were 12 C or higher. Six days of high temperature also resulted in a reduction in endosperm FW, nuclei number and a delay in the peak of mitotic index. DNA endoreduplication occurred in the kernels exposed to this treatment, although the magnitude was severely reduced compared with the control kernels. Nuclear DNA content was highly correlated (r = 0·93) with kernel FW, suggesting an important role of DNA endoreduplication in determining endosperm FW. The data suggest that high temperature during endosperm cell division exerted negative effects on DNA endoreduplication by dramatically reducing the nuclei number, leaving fewer nuclei available for DNA endoreduplication. However, the data also suggest that prolonged exposure to high temperature restricts entry of mitotic cells into the endoreduplication phase of the cell cycle.     

Starch Synthesis and Programmed Cell Death during Endosperm Development in Triticale (x Triticosecale Wittmack)

Triticale (x Triticosecale Wittmack) grains synthesize and accumulate starch as their main energy source.Starch accumulation rate and synthesis activities of ADP-glucose pyrophosphorylase, soluble starch synthases, granule-bound starch synthase and starch-branching enzyme showed similar pattern of unimodal curves during endosperm development. There was no significant difference in activity of the starch granule-bound protein isolated from total and separated starch granules at different developmental stages after anthesis in triticale. Evans Blue staining and analysis of DNA fragmentation indicated that cells of triticale endosperm undergo programmed cell death during its development. Dead cells within the endosperm were detected at 6 d post anthesis (DPA), and evidence of DNA fragmentation was first observed at 21 DPA. The period between initial detection of PCD to its rapid increase overlapped with the key stages of rapid starch accumulation during endosperm development. Cell death occurred stochastically throughout the whole endosperm, meanwhile, the activities of starch biosynthetic enzymes and the starch accumulation rate decreased in the late stages of grain filling. These results suggested that the timing and progression of PCD in triticale endosperm may interfere with starch synthesis and accumulation.     

Starch Synthesis and Programmed Cell Death during Endosperm Development in Triticale(× Triticosecale Wittmack)

Triticale(× Triticosecale Wittmack) grains synthesize and accumulate starch as their main energy source.Starch accumulation rate and synthesis activities of ADP-glucose pyrophosphorylase,soluble starch synthases,granule-bound starch synthase and starch-branching enzyme showed similar pattern of unimodal curves during endosperm development.There was no significant difference in activity of the starch granule-bound protein isolated from total and separated starch granules at different developmental stages after anthesis in triticale.Evans Blue staining and analysis of DNA fragmentation indicated that cells of triticale endosperm undergo programmed cell death during its development.Dead cells within the endosperm were detected at 6 d post anthesis(DPA),and evidence of DNA fragmentation was first observed at 21 DPA.The period between initial detection of PCD to its rapid increase overlapped with the key stages of rapid starch accumulation during endosperm development.Cell death occurred stochastically throughout the whole endosperm,meanwhile,the activities of starch biosynthetic enzymes and the starch accumulation rate decreased in the late stages of grain filling.These results suggested that the timing and progression of PCD in triticale endosperm may interfere with starch synthesis and accumulation.     

Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase

Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme.     

Growth and composition of maize kernels cultured in vitro with varying supplies of carbon and nitrogen

This study employed in vitro seed culture to determine how C and N supply influence the growth (i.e. starch accumulation) and protein composition of maize (Zea mays L.) endosperm. Immature kernels were grown to maturity on liquid medium containing various concentrations of C (sucrose at 234 millimolar [low] and 468 millimolar [high]) and N (amino acid mixture ranging in N from 0 to 144 millimolar). Low C supply limited starch, but not N, accumulation in the endosperm. With high C, endosperm starch and protein content increased concomitantly as N supply increased from 0 to 13.4 millimolar. Endosperm growth was unaffected by additional N until concentrations exceeding approximately 72 millimolar reduced starch accumulation. A similar inhibition of starch deposition occurred with lower N concentrations when kernels were grown with low C. Endosperm total N content reached a point of saturation with approximately 36 millimolar N in the medium, regardless of C supply. Zein synthesis in the endosperm responded positively across all N levels, while glutelin content remained static and albumin/globulin proteins were reduced in amount when N supply was greater than 36 millimolar. A reciprocal, inverse relationship was observed in mature endosperm tissue between the concentrations of free amino acids and soluble sugars. Our data suggest that under N stress starch and protein accumulation in the endosperm are interdependent, at least in appearance, but are independent otherwise.