Immature Endosperm Research Articles

Control of seed and root development by WIPK-activated transcription factor, NtWIF in tobacco plants

Mitogen-activated protein kinase (MAPK) is one of the critical mediators of signaling systems by constituting phosphorylation pathways together with upstream kinases. Despite the importance, few direct targets have so far been identified, making it difficult to understand the whole features of so called MAPK cascade. Notably, a novel transcription factor, which was recently identified from tobacco, is activated upon phosphorylation by wound-induced protein kinase (WIPK), a typical MAPK in tobacco plants, and designated as NtWIF (Nicotiana tobacum WIPK interacting factor). To identify genes that are controlled by NtWIF, a microarray screening was performed using NtWIF overexpressing transgenic tobacco, and a set of affected clones were isolated. Approximately 28% of them were related to defense, confirming the critical role of WIPK in stress response. In addition, 15% of the clones were related to differentiation, suggesting pleiotrophic effects of NtWIF. When NtWIF was overexpressed or silenced by RNAi in tobacco plants, abnormal seed development and root growth were observed. Seeds of RNAi lines exhibited immature endosperm, resulting in germination failure. Root growth was retarded in both transgenic seedlings due to abnormal cell numbers in meristem. These results implicated the participation of phytohormones, of which function might be controlled by NtWIF. Since NtWIF possesses a B3-DNA binding motif, which specifically recognizes auxin-responsive elements found in many auxin-responsive genes, the present finding suggested that WIPK/NtWIF functions not only in stress response but also in developmental process through hormonal control.

THE PROGRAMMED CELL DEATH, RESCUING CULTURE AND POLYPLOID PLANTLET ESTABLISHMENT IN ENDOSPERM CULTURE OF SPIDER LILIES (LYCORIS SPP.)

ISHS V International Symposium on In Vitro Culture and Horticultural Breeding THE PROGRAMMED CELL DEATH, RESCUING CULTURE AND POLYPLOID PLANTLET ESTABLISHMENT IN ENDOSPERM CULTURE OF SPIDER LILIES (LYCORIS SPP.)

Evaluation of in vitro methods to define the role of the cob and pedicel-placento-chalazal tissues in the amino acid supply to the developing maize endosperm

Two in vitro methods that mimic the in vivo (field conditions) endosperm development of the Illinois High (IHP) and Illinois Low Protein (ILP) maize strains were evaluated. For the first method, immature endosperms, 9 days after pollination (DAP), were grown for 5 days on solid media containing a source of inorganic nitrogen (N) in the presence or absence of glutamine. On both culture media, the ILP endosperms accumulated more starch than the IHP endosperms. The same occurred with in vivo grown endosperms. In vitro ILP had the same zein content as IHP, reaching higher values than that observed at the same stage in vivo. The chromosome endoreduplication analysis of IHP and ILP endosperms in vitro cultured with this method, exhibited a mean ploidy level significantly lower than controls at the same stage for both strains. For the second method, ears were harvested at 4 and 9 DAP, cut into cob-blocks and cultured until 25 DAP. Both IHP and ILP kernels grown in vitro showed, for the specific strain, a phenotype similar to that observed in kernels grown at the same stage in field conditions. An experiment including 14 C -glutamine in the culture media of cob-blocks suggested that the cob and pedicel-placento-chalazal (PPCh) tissues may be involved in determining the ILP phenotype.

Evaluation of expression cassettes in developing rice endosperm using a transient expression assay

In order to evaluate rapidly the potential of various expression cassettes for transgene expression in rice grain, a transient expression assay was developed using rice immature endosperms. The promoters of several seed storage protein genes including the rice glutelins ( Gt3, GluB-1, GluB-2), rice prolamins ( PG5a, RP6), rice α-globulin ( Glb) and wheat glutenin ( Bx7), were examined for their expression levels in rice immature endosperms isolated from the caryopses at 7–9 days after pollination. In this promoter comparison, the wheat Bx7 promoter showed the highest transcription activity, followed by the promoters of the Glb, GluB-2, and Gt3 genes. Based on this result, further studies were conducted using the Glb and Bx7 promoters. We examined the effects of the addition of a second promoter fragment, the alfalfa mosaic virus (AMV) 5′untranslated leader sequence (UTL), and various introns and terminators from different genes. Although the addition of a second promoter fragment and the AMV 5′UTL had no effect on GUS expression levels, the insertion of the rice waxy first intron increased GUS expression of the Glb and Bx7 constructs 3 and 6-fold, respectively. The maize adh1 first intron also showed a 2–3-fold increase in the GUS expression levels of both the Glb and Bx7 reporter gene constructs. A comparison of terminator sequences revealed the nopaline synthase gene terminator from Agrobacterium and the rice waxy gene terminator were the most effective in contributing to elevated expression levels.

Antisense waxy genes with highly active promoters effectively suppress waxy gene expression in transgenic rice.

To regulate Waxy (Wx) gene expression by introducing antisense genes, we connected the 2.3 kb Wx cDNA having 450 bp of the Wx first intron in reverse orientation to rice Wx and maize alcohol dehydrogenase1 (Adh1) promoters and used these constructs to transform rice plants. Of 10 independent transgenic lines analysed, four lines showed various degrees of reduction in amylose and WAXY (WX) protein levels in the endosperm. In two transgenic lines, complete absence of amylose was observed which made the seeds opaque white like glutinous rice (amylose-deficient waxy (wx) mutant). In one of the transgenic lines, A1 line, the presence of the antisense Wx gene cosegregated with reduction of amylose content in the endosperm. In the same line, a reduction in the level of endogenous Wx mRNA was observed in immature endosperm. Interestingly, this reduction was observed only with mature spliced transcripts but not with unspliced transcripts. Reduced amylose synthesis was also observed in pollen grains of four transgenic lines. These results suggest that integrated antisense Wx gene caused a reduction in amylose synthesis in endosperms and pollen grains of transgenic rice carrying the antisense Wx cDNA. These results indicate that manipulation of starch and other carbohydrates in rice grain is possible using antisense genes.

Production of triploid plants of mulberry (Morus alba L) by endosperm culture.

A continuously growing callus was obtained from immature endosperm of Morus alba L Cv S-36 cultured on Murashige and Skoog medium containing 5 μm 2,4-dichlorophenoxyacetic acid. Shoot buds were produced when the callus was subcultured on a medium containing a cytokinin or a cytokinin and 1-naphthaleneacetic acid (NAA). The maximum number of shoots was formed on the medium containing thidiazuron (1 μM), or benzylaminopurine (5 μM) and NAA (1 μM). Shoots were multiplied by forced axillary branching and rooted in vitro. Endosperm-derived plants were established in soil. Each of the ten plants examined cytologically was triploid (3 n=42).

Identification of a DNA-binding factor that recognizes an alpha-coixin promoter and interacts with a Coix Opaque-2 like protein.

Transient expression and electrophoretic mobility shift assay were used to investigate the cis elements and the DNA-binding proteins involved in the regulation of expression of a 22 kDa zein-like alpha-coixin gene. A set of unidirectional deletions was generated in a 962 bp fragment of the alpha-coixin promoter that had been previously fused to the reporter gene GUS. The constructs were assayed by transient expression in immature maize endosperm. There was no significant decrease in GUS activity as deletions progressed from -1084 to -238. However, deletion from -238 to -158, which partially deleted the O2c box, resulted in a dramatic decrease in GUS activity emphasizing the importance of the O2 box in the quantitative expression of the gene. The -238 promoter fragment interacted with Coix endosperm nuclear proteins to form 5 DNA-protein complexes, C1-C5, as detected by EMSA. The same retarded complexes were observed when the -158 promoter fragment was used in the binding reactions. Reactions with nuclear extracts isolated from Coix endosperms harvested from 6 to 35 days after pollination revealed that the 5 DNA-protein complexes that interact with the alpha-coixin promoter are differentially assembled during seed development. Deletion analysis carried out on the -238/ATG promoter fragment showed that a 35 bp region from -86 to -51 is essential for the formation of the complexes observed. When nuclear extracts were incubated with an antiserum raised against the maize Opaque-2 protein, the formation of 4 complexes, C1, C3, C4 and C5, was prevented indicating that an Opaque-2 like protein participates in the formation of those complexes. Complex C2 was not affected by the addition of the O2 antibody, suggesting the existence of a novel nuclear factor, CBF1, that binds to the promoter and makes protein-protein associations with other proteins present in Coix endosperm nuclei.

Somatic embryogenesis and regeneration of triploid plants in endosperm cultures of Acacia nilotica

Immature endosperm of Acacia nilotica formed a nodular callus on MS medium supplemented with 2,4-D, BAP and CH. In the third passage on this medium, in the dark, the callus differentiated somatic embryos. The embryos germinated on MS only after 15 d pre-treatment on modified MS medium in which major salts were replaced by those of major salts of B5 medium and supplemented with glutamine, CH and CW. Triploid nature of the somatic embryos was confirmed by Feulgen cytophotometry.

Lysophosphatidic acid acyltransferase from immature coconut endosperm having medium chain length substrate specificity

Immature endosperm of coconut ( Cocos nucifera) contains a membrane-bound lysophosphatidic acid acyltransferase (LPAAT) having medium chain length substrate specificity appropriate to the biosynthesis of coconut oil. Acyl-CoAs containing 10:0, 12:0 and 14:0 acyl groups are the preferred acyl-donor substrates; acyl-ACPs are not utilized. There is slight preference for 12:0-lysophosphatidic acid (LPA) over 18:1-LPA as acceptor substrate. Treatment of the active membrane fraction with 2.25% (w/v) CHAPS, at a detergent:protein ratio of 48:1 (w/v), in the presence of 1M NaCl solubilized the enzyme in high yield. Solubilization was evidenced by three independent criteria, namely, failure of the activity to sediment at high centrifugal force, behaviour of the activity as a globular protein of apparent M r 44000 in size-exclusion chromatography, and partial resolution of the activity from many of the membrane proteins on the size-exclusion column. Optimal restoration of LPAAT activity after solubilization required the addition of detergent-treated phospholipids, in addition to a lowering of the detergent and NaCl concentrations.

Phytelephas aequatorialis (arecaceae) in human and animal nutrition

Field observations revealed the consumption of several parts of the vegetable ivory (“tagua”) palm not previously reported to be eaten by humans or animals. The whole male inflorescence provides cattle with a fodder nutritionally similar to ryegrass, while the flower clusters provide humans with 102 Kcal/100 g, about four times the caloric density of cauliflower or broccoli. The central mesocarp is similar in composition and caloric density to other fruits and is a comparatively rich source of calcium (116 mg/100 g), potassium (841 mg/100 g), and zinc (1.3 mg/100 g). The interior mesocarp, with 22% fat, is a high caloric density (288 Kcal/100 g) fodder for chickens and is rich in linoleic acid (21%). The immature endosperm, eaten as a snack, is of negligible importance in human nutrition.

In vitro culture of coconut endosperm: callus induction and its fatty acids

Successful induction of callus from coconut endosperm was achieved by using the tissue situated near the micropylar end of a young fruit. For initiation of callus, a high concentration of auxin (20 to 100 ppm) was added to the basal medium containing activated charcoal. Subcultured callus showed a 40-fold increase during culture of three months. Based on the analysis of fatty acid composition, the maturation of endosperm was characterized by an increase in short chain fatty acids (C8, C10, C12, C14)and a decrease in long chain fatty acids (C16, C18: 1, C18: 2). In developing endosperms, proportion of short chain fatty acids was higher in lipids of the antipodal than those of other regions. In the final stage of maturation, around 82% of total fatty acids was short chain fatty acids, while the proportion of long chain fatty acids decreased up to 16%. The fatty acid composition of callus subcultured for six months was comparable to that of the immature endosperm. Lipids were accumulated in callus as globular bodies.

Functional analysis of the regulatory region of a zein gene in transiently transformed protoplasts.

The transcription of zein genes in maize is tissue-specific and developmentally regulated. The 5' regulatory region of many zein genes contains two promoters, P1 and P2, lying approximately 1000 bases apart. The promoter/enhancer activity of various fragments of the two promoter regions of the zein gene E19 have been analysed by means of transient expression experiments. The results indicate that the various regions differentially affect the expression of the GUS reporter gene activity in protoplasts from tobacco leaves, maize immature endosperms and in vitro endosperm cell cultures. In tobacco protoplasts only the proximal promoter region, P2, activates GUS expression, while in endosperm culture cells only the distant promoter, P1, gives significant activity. The P1 region, both in direct and opposite orientation, stimulates a low level of GUS expression in protoplasts from immature endosperms.

Synthesis and deposition of coixin in seeds of Coix lacryma-jobi

The synthesis and assembly of prolamins into protein bodies of Coix lacryma-jobi seeds were investigated. Coixins, the Coix prolamins, are grouped into two distinct classes namely α- and γ-coixins. Alpha-coixins are constituted by four size classes, while γ-coixins comprise only one molecular weight class. SDS-PAGE and western blot analysis of prolamins extracted from endosperm during seed development showed that α-coixins are synthesized at earlier developmental stages than γ-coixin. In vitro translation of polysomal RNA attached to protein bodies isolated from mid-maturation endosperm showed that these polyribosomes are highly enriched in coixin messages. Polysomal RNAs isolated from all developmental stages were electrophoresed and probed with cDNA clones representing α- and γ-coixins. The results confirmed the earlier expression of α-coixins and also demonstrated that coixin RNA accumulation in the endosperm occurs mainly at seed mid maturation. Protein bodies isolated from immature endosperm contained all coixin components as determined by SDS-PAGE and western blot analyses. Immunocytochemical analysis by electron and light microscopy revealed that the coixin components are spread all over the protein bodies. The protein bodies are localized in the starchy endosperm cells filling the spaces left by the starch granules. They are surrounded by continuous membranes and are larger than the protein bodies described for maize.

Stable endosperm cell suspension cultures from wild-type and opaque-2 maize

Stable cell suspension cultures have been established from immature endosperms of A69Y wild-type and opaque-2 maize (Zea mays L.). Cultured cells are capable of storage protein (zein) synthesis and accumulation throughout the growth period. Electrophoretic patterns of zeins show, for opaque-2 cells, the preferential inhibition of the accumulation of 22 kDa peptides typical of the mutation. Viable protoplasts, able to regenerate cell walls, as well as to divide and to express foreign DNA in transient expression experiments, can be obtained with high yields from cultures of both genotypes.

Caffeine metabolism in Coffea arabica and other species of coffee

High performance liquid chromatography has been used to measure the quantities of caffeine, theobromine, and theophylline in aqueous extracts of endosperm from immature and mature fruits of Coffea arabica and six other species of Coffea. Caffeine was the alkaloid present in largest amounts and, with one exception, in concentrations that were broadly similar in immature and mature fruit. The highest concentrations of caffeine were found in C. canephora at 35.1 and 24.5 mg g −1, respectively, in immature and mature endosperm. The lowest concentrations were in C. bengalensis, where caffeine was not detected in extracts from mature fruit. [8- 3H]Caffeine was metabolised relatively slowly by immature endosperm of C. arabica and C. canephora. In contrast, C. dewevrei, C. eugenioides, C. stenophylla, C. salvatrix and C. bengalensis all appeared to metabolise [8- 3H]caffeine much more rapidly, as the percentage recovery of the applied label was much lower and there was more extensive incorporation of radioactivity into theobromine, theophylline, 3-methylxanthine and two unidentified polar metabolites. The endogenous caffeine concentrations and the metabolism data indicate that there may be marked differences in the rate of turnover of caffeine in the various species of Coffea. Potential sources of material for the production of naturally decaffeinated coffee are discussed.

Analysis of in vivo and in vitro grown endosperms of high and low protein strains of maize

The aim of this research was to examine the effect of C and N supply on growth and protein composition of maize ( Zea mays L.) endosperm. Immature endosperms of the Illinois High and Low Protein (IHP and ILP) strains were grown for 5, 8 and 12 days on a solid medium containing different sucrose to asparagine ratios. A comparison between endosperms of IHP and ILP strains grown to maturity in vitro and in field conditions (in vivo) was also reported. The results showed that the ILP strain cultured in vitro accumulated a greater amount of dry matter per endosperm than the IHP strain; this was also observed in in vivo experiments. The accumulation of total nitrogen per endosperm followed a different trend when the in vivo and the in vitro conditions were compared. In vivo IHP was more efficient than ILP in assimilating nitrogen. The reverse was observed in vitro; there the ILP strain was always capable of accumulating higher amounts of N per endosperm than IHP at all stages of development considered and on all culture media. IHP endosperms, during development in vivo and in vitro, synthesized both the 22 kDa fractions of zeins. While ILP endosperms grown in vivo were much less efficient in accumulating the 22 kDa fraction of zeins, these latter endosperms cultured in vitro were capable of restoring normal synthesis of the 22 kDa zein fraction. Our data suggest that the expression of endosperm phenotype was affected by nitrogen substrates supplied to the seed.

Plant tissue culture of Secale: A review

Plant tissue culture of rye employs different parts of plant bodies originating from various stages of rye ontogenesis. For the culture initiation mainly diploid and after that tetraploid forms were used, however, haploids and triploids were subject of investigation. The development of plant regeneration via somatic embryogenesis or organogenesis required the using of numerous basal media with various plant growth hormones. Haploidization appeared to be the most difficult problem and only interspecies hybridization helped to overcome this problem. Wide sexual intergenera and interspecies hybridization in cereals require the development of proembryo and immature embryo culture system. Using the ‘nurse culture’ basing on immature endosperm resulted in getting fully formed rye plants. There is no progress in somatic cell genetic manipulation of rye because of lack of plant regeneration system in mesophyll green leaf and suspension protoplast cultures.

A rice glutelin gene family — a major type of glutelin mRNAs can be divided into two classes

Rice glutelin cDNA clones were isolated from a rice cDNA library constructed from immature endosperm. The isolated glutelin cDNA clones could be divided into two classes by restriction site polymorphism and sequence. They encoded polypeptides containing the same number of amino acids including a 24 amino acid signal peptide, a 282 amino acid acidic subunit and a 193 amino acid basic subunit. The coding regions were 95% homologous to each other at the nucleotide and amino acid levels. Southern hybridization analysis revealed that there were four or five copies of glutelin genes per haploid rice genome.

Isolation and characterization of an amyloplast envelope‐enriched fraction from immature maize endosperm

Gardner, H. W., Miernyk, J. A., Christianson, D. D. and Khoo, U. 1987. Isolation and characterization of an amyloplast envelope‐enriched fraction from immature maize endosperm.A 10000–100000 g pellet obtained by centrifugation of homogenates from immature (25 days after pollination) de‐embryonated maize (Zea mays L., cv. W64A‐normal and a typical hybrid) kernels was further fractionated by sedimentation on discontinuous sucrose density gradients. Particles with the highest carotenoid content (0.68% by weight carotenoids based upon total lipid) sedimented at densities of 1.083‐1.106 g ml‐1, coincident with the plastid envelope marker enzyme, galacto‐syltransferase (EC 2.4.1.46). Lipids extracted from the carotenoid‐rich fraction were mainly digalactosyldiacylglycerols, monogalactosyldiacylglycerols, phosphatidylcholines, phosphatidylinositols and phosphatidylglycerols, in order of molar abundance. With increasing particle density (>1.106 g ml1) the phospholipid and neutral lipid content increased, and the proportion of carotenoids and galactolipids decreased. Electron micrographs of the carotenoid‐rich fraction revealed vesicles ranging in size from < 0.1 to 0.5 um, as well as smaller granular membranes. The carotenoid‐rich membrane fraction was progressively more difficult to isolate as the endosperm matured, and freezing the immature endosperm prevented subsequent isolation. The lipid and enzyme composition and ultrastructural characteristics of the isolated fraction suggest that it is composed of amyloplast envelope vesicles.

In maize, glutelin-2 and low molecular weight zeins are synthesized by membrane-bound polyribosomes and translocated into microsomal membranes

Experiments to establish the site of biosynthesis and the possible translocation into microsomes of glutelins-2 (28 kD G2) and low molecular weight zeins (10, 14, 15 kD Z2) have been carried out. Free and membrane-bound polyribosomes as well as microsomal membranes were isolated from immature endosperms of W64A Zea mays L. In vitro translation studies were carried out in the presence and in the absence of membranes using [(35)S]-methionine or [(35)S]-cysteine as precursors. Cell-free translation products were characterized by electrophoretic mobility, solubility and antigenic properties. The results obtained indicate that 28 kD G2 and low molecular weight zeins are primarily synthesized on membrane-bound polysomes. From experiments using proteinase K as a probe, we also conclude that these proteins are translocated into microsomes where they accumulate. The translocated and pre-28 kD G2 proteins do not present changes in the apparent molecular weight. However we show that there are differences in their isoelectric points, a fact that indicates the existence of 28 kD G2 processing.