Showing papers on "Endosperm published in 2013"

ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination.

Abstract: Dormancy is an adaptive trait that enables seed germination to coincide with favorable environmental conditions. It has been clearly demonstrated that dormancy is induced by abscisic acid (ABA) during seed development on the mother plant. After seed dispersal, germination is preceded by a decline in ABA in imbibed seeds, which results from ABA catabolism through 8'-hydroxylation. The hormonal balance between ABA and gibberellins (GAs) has been shown to act as an integrator of environmental cues to maintain dormancy or activate germination. The interplay of ABA with other endogenous signals is however less documented. In numerous species, ethylene counteracts ABA signaling pathways and induces germination. In Brassicaceae seeds, ethylene prevents the inhibitory effects of ABA on endosperm cap weakening, thereby facilitating endosperm rupture and radicle emergence. Moreover, enhanced seed dormancy in Arabidopsis ethylene-insensitive mutants results from greater ABA sensitivity. Conversely, ABA limits ethylene action by down-regulating its biosynthesis. Nitric oxide (NO) has been proposed as a common actor in the ABA and ethylene crosstalk in seed. Indeed, convergent evidence indicates that NO is produced rapidly after seed imbibition and promotes germination by inducing the expression of the ABA 8'-hydroxylase gene, CYP707A2, and stimulating ethylene production. The role of NO and other nitrogen-containing compounds, such as nitrate, in seed dormancy breakage and germination stimulation has been reported in several species. This review will describe our current knowledge of ABA crosstalk with ethylene and NO, both volatile compounds that have been shown to counteract ABA action in seeds and to improve dormancy release and germination.

The ubiquitin receptor DA1 interacts with the E3 ubiquitin ligase DA2 to regulate seed and organ size in Arabidopsis.

Abstract: Seed size in higher plants is determined by the coordinated growth of the embryo, endosperm, and maternal tissue. Several factors that act maternally to regulate seed size have been identified, such as AUXIN RESPONSE FACTOR2, APETALA2, KLUH, and DA1, but the genetic and molecular mechanisms of these factors in seed size control are almost totally unknown. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligase ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to regulate the final size of seeds in Arabidopsis thaliana. Here, we describe another RING-type protein with E3 ubiquitin ligase activity, encoded by DA2, which regulates seed size by restricting cell proliferation in the maternal integuments of developing seeds. The da2-1 mutant forms large seeds, while overexpression of DA2 decreases seed size of wild-type plants. Overexpression of rice (Oryza sativa) GRAIN WIDTH AND WEIGHT2, a homolog of DA2, restricts seed growth in Arabidopsis. Genetic analyses show that DA2 functions synergistically with DA1 to regulate seed size, but does so independently of EOD1. Further results reveal that DA2 interacts physically with DA1 in vitro and in vivo. Therefore, our findings define the genetic and molecular mechanisms of three ubiquitin-related proteins DA1, DA2, and EOD1 in seed size control and indicate that they are promising targets for crop improvement.

Structure and Composition

Abstract: Seeds are very diverse in their shape and size. In the mature state they contain an embryo, the next generation of plant, surrounded by a protective structure (the seed and/or fruit coat) and, in species in which the nutritive reserves are not stored within the cotyledons, by an alternative storage tissue (endosperm, perisperm, or megagametophyte). Most seeds contain large and characteristic quantities of polymeric reserves. The major ones are carbohydrates, oils, and proteins, with minor amounts of phosphate-rich phytin. Starch, a polymer of glucose, is the most common form of stored carbohydrate, contained within cytoplasmic starch granules; less common are the hemicelluloses, stored in secondary cell walls, usually as mannan polymers. Oils are triacylglycerols, each composed of glycerol and three fatty acids that are specific to the oil; these are present within oil bodies. Storage proteins, of which there are three types, albumins, globulins, and prolamins, are sequestered in protein storage vacuoles. These reserves are vital components of human and animal diets, and their production in crops is a basis of agriculture.

OsbZIP58, a basic leucine zipper transcription factor, regulates starch biosynthesis in rice endosperm

Abstract: Starch composition and the amount in endosperm, both of which contribute dramatically to seed yield, cooking quality, and taste in cereals, are determined by a series of complex biochemical reactions. However, the mechanism regulating starch biosynthesis in cereal seeds is not well understood. This study showed that OsbZIP58, a bZIP transcription factor, is a key transcriptional regulator controlling starch synthesis in rice endosperm. OsbZIP58 was expressed mainly in endosperm during active starch synthesis. osbzip58 null mutants displayed abnormal seed morphology with altered starch accumulation in the white belly region and decreased amounts of total starch and amylose. Moreover, osbzip58 had a higher proportion of short chains and a lower proportion of intermediate chains of amylopectin. Furthermore, OsbZIP58 was shown to bind directly to the promoters of six starch-synthesizing genes, OsAGPL3, Wx, OsSSIIa, SBE1, OsBEIIb, and ISA2, and to regulate their expression. These findings indicate that OsbZIP58 functions as a key regulator of starch synthesis in rice seeds and provide new insights into seed quality control.

Comparative Transcriptome Analysis of Three Oil Palm Fruit and Seed Tissues That Differ in Oil Content and Fatty Acid Composition

Abstract: Oil palm (Elaeis guineensis) produces two oils of major economic importance, commonly referred to as palm oil and palm kernel oil, extracted from the mesocarp and the endosperm, respectively. While lauric acid predominates in endosperm oil, the major fatty acids (FAs) of mesocarp oil are palmitic and oleic acids. The oil palm embryo also stores oil, which contains a significant proportion of linoleic acid. In addition, the three tissues display high variation for oil content at maturity. To gain insight into the mechanisms that govern such differences in oil content and FA composition, tissue transcriptome and lipid composition were compared during development. The contribution of the cytosolic and plastidial glycolytic routes differed markedly between the mesocarp and seed tissues, but transcriptional patterns of genes involved in the conversion of sucrose to pyruvate were not related to variations for oil content. Accumulation of lauric acid relied on the dramatic up-regulation of a specialized acyl-acyl carrier protein thioesterase paralog and the concerted recruitment of specific isoforms of triacylglycerol assembly enzymes. Three paralogs of the WRINKLED1 (WRI1) transcription factor were identified, of which EgWRI1-1 and EgWRI1-2 were massively transcribed during oil deposition in the mesocarp and the endosperm, respectively. None of the three WRI1 paralogs were detected in the embryo. The transcription level of FA synthesis genes correlated with the amount of WRI1 transcripts and oil content. Changes in triacylglycerol content and FA composition of Nicotiana benthamiana leaves infiltrated with various combinations of WRI1 and FatB paralogs from oil palm validated functions inferred from transcriptome analysis.

Transcriptional dynamics of two seed compartments with opposing roles in Arabidopsis seed germination

Abstract: Seed germination is a critical stage in the plant life cycle and the first step toward successful plant establishment. Therefore, understanding germination is of important ecological and agronomical relevance. Previous research revealed that different seed compartments (testa, endosperm, and embryo) control germination, but little is known about the underlying spatial and temporal transcriptome changes that lead to seed germination. We analyzed genome-wide expression in germinating Arabidopsis (Arabidopsis thaliana) seeds with both temporal and spatial detail and provide Web-accessible visualizations of the data reported (vseed.nottingham.ac.uk). We show the potential of this high-resolution data set for the construction of meaningful coexpression networks, which provide insight into the genetic control of germination. The data set reveals two transcriptional phases during germination that are separated by testa rupture. The first phase is marked by large transcriptome changes as the seed switches from a dry, quiescent state to a hydrated and active state. At the end of this first transcriptional phase, the number of differentially expressed genes between consecutive time points drops. This increases again at testa rupture, the start of the second transcriptional phase. Transcriptome data indicate a role for mechano-induced signaling at this stage and subsequently highlight the fates of the endosperm and radicle: senescence and growth, respectively. Finally, using a phylotranscriptomic approach, we show that expression levels of evolutionarily young genes drop during the first transcriptional phase and increase during the second phase. Evolutionarily old genes show an opposite pattern, suggesting a more conserved transcriptome prior to the completion of germination.

Brassinosteroid Regulates Seed Size and Shape in Arabidopsis

Abstract: Seed development is important for agriculture productivity. We demonstrate that brassinosteroid (BR) plays crucial roles in determining the size, mass, and shape of Arabidopsis (Arabidopsis thaliana) seeds. The seeds of the BR-deficient mutant de-etiolated2 (det2) are smaller and less elongated than those of wild-type plants due to a decreased seed cavity, reduced endosperm volume, and integument cell length. The det2 mutant also showed delay in embryo development, with reduction in both the size and number of embryo cells. Pollination of det2 flowers with wild-type pollen yielded seeds of normal size but still shortened shape, indicating that the BR produced by the zygotic embryo and endosperm is sufficient for increasing seed volume but not for seed elongation, which apparently requires BR produced from maternal tissues. BR activates expression of SHORT HYPOCOTYL UNDER BLUE1, MINISEED3, and HAIKU2, which are known positive regulators of seed size, but represses APETALA2 and AUXIN RESPONSE FACTOR2, which are negative regulators of seed size. These genes are bound in vivo by the BR-activated transcription factor BRASSINAZOLE-RESISTANT1 (BZR1), and they are known to influence specific processes of integument, endosperm, and embryo development. Our results demonstrate that BR regulates seed size and seed shape by transcriptionally modulating specific seed developmental pathways.

Identification and characterization of the zinc-regulated transporters, iron-regulated transporter-like protein (ZIP) gene family in maize.

Abstract: Zinc (Zn) and iron (Fe) are essential micronutrients for plant growth and development, their deficiency or excess severely impaired physiological and biochemical reactions of plants. Therefore, a tightly controlled zinc and iron uptake and homeostasis network has been evolved in plants. The Zinc-regulated transporters, Iron-regulated transporter-like Proteins (ZIP) are capable of uptaking and transporting divalent metal ion and are suggested to play critical roles in balancing metal uptake and homeostasis, though a detailed analysis of ZIP gene family in maize is still lacking. Nine ZIP-coding genes were identified in maize genome. It was revealed that the ZmZIP proteins share a conserved transmembrane domain and a variable region between TM-3 and TM-4. Transiently expression in onion epidermal cells revealed that all ZmZIP proteins were localized to the endoplasmic reticulum and plasma membrane. The yeast complementation analysis was performed to test the Zn or Fe transporter activity of ZmZIP proteins. Expression analysis showed that the ZmIRT1 transcripts were dramatically induced in response to Zn- and Fe-deficiency, though the expression profiles of other ZmZIP changed variously. The expression patterns of ZmZIP genes were observed in different stages of embryo and endosperm development. The accumulations of ZmIRT1 and ZmZIP6 were increased in the late developmental stages of embryo, while ZmZIP4 was up-regulated during the early development of embryo. In addition, the expression of ZmZIP5 was dramatically induced associated with middle stage development of embryo and endosperm. These results suggest that ZmZIP genes encode functional Zn or Fe transporters that may be responsible for the uptake, translocation, detoxification and storage of divalent metal ion in plant cells. The various expression patterns of ZmZIP genes in embryo and endosperm indicates that they may be essential for ion translocation and storage during differential stages of embryo and endosperm development. The present study provides new insights into the evolutionary relationship and putative functional divergence of the ZmZIP gene family during the growth and development of maize.

Development of Low Phytate Rice by RNAi Mediated Seed-Specific Silencing of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase Gene (IPK1)

Abstract: Phytic acid (InsP6) is considered to be the major source of phosphorus and inositol phosphates in most cereal grains. However, InsP6 is not utilized efficiently by monogastric animals due to lack of phytase enzyme. Furthermore, due to its ability to chelate mineral cations, phytic acid is considered to be an antinutrient that renders these minerals unavailable for absorption. In view of these facts, reducing the phytic acid content in cereal grains is a desired goal for the genetic improvement of several crops. In the present study, we report the RNAi-mediated seed-specific silencing (using the Oleosin18 promoter) of the IPK1 gene, which catalyzes the last step of phytic acid biosynthesis in rice. The presence of the transgene cassette in the resulting transgenic plants was confirmed by molecular analysis, indicating the stable integration of the transgene. The subsequent T4 transgenic seeds revealed 3.85-fold down-regulation in IPK1 transcripts, which correlated to a significant reduction in phytate levels and a concomitant increase in the amount of inorganic phosphate (Pi). The low-phytate rice seeds also accumulated 1.8-fold more iron in the endosperm due to the decreased phytic acid levels. No negative effects were observed on seed germination or in any of the agronomic traits examined. The results provide evidence that silencing of IPK1 gene can mediate a substantial reduction in seed phytate levels without hampering the growth and development of transgenic rice plants.

Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm

Abstract: Arabidopsis thaliana endosperm, a transient tissue that nourishes the embryo, exhibits extensive localized DNA demethylation on maternally inherited chromosomes. Demethylation mediates parent-of-origin–specific (imprinted) gene expression but is apparently unnecessary for the extensive accumulation of maternally biased small RNA (sRNA) molecules detected in seeds. Endosperm DNA in the distantly related monocots rice and maize is likewise locally hypomethylated, but whether this hypomethylation is generally parent-of-origin specific is unknown. Imprinted expression of sRNA also remains uninvestigated in monocot seeds. Here, we report high-coverage sequencing of the Kitaake rice cultivar that enabled us to show that localized hypomethylation in rice endosperm occurs solely on the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small class II (cut-and-paste) transposable elements, those in endosperm are more uniformly derived, including sequences from other transposon classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that localized hypomethylation of maternal endosperm DNA is conserved in flowering plants.

GIANT EMBRYO encodes CYP78A13, required for proper size balance between embryo and endosperm in rice.

Abstract: Among angiosperms there is a high degree of variation in embryo/endosperm size in mature seeds. However, little is known about the molecular mechanism underlying size control between these neighboring tissues. Here we report the rice GIANT EMBRYO (GE) gene that is essential for controlling the size balance. The function of GE in each tissue is distinct, controlling cell size in the embryo and cell death in the endosperm. GE, which encodes CYP78A13, is predominantly expressed in the interfacing tissues of the both embryo and endosperm. GE expression is under negative feedback regulation; endogenous GE expression is upregulated in ge mutants. In contrast to the loss-of-function mutant with large embryo and small endosperm, GE overexpression causes a small embryo and enlarged endosperm. A complementation analysis coupled with heterofertilization showed that complementation of ge mutation in either embryo or endosperm failed to restore the wild-type embryo/endosperm ratio. Thus, embryo and endosperm interact in determining embryo/endosperm size balance. Among genes associated with embryo/endosperm size, REDUCED EMBRYO genes, whose loss-of-function causes a phenotype opposite to ge, are revealed to regulate endosperm size upstream of GE. To fully understand the embryo-endosperm size control, the genetic network of the related genes should be elucidated.

Integration of epigenetic and genetic controls of seed size by cytokinin in Arabidopsis

Abstract: The development of seeds in flowering plants is placed under complex interactions between maternal tissues, the embryo, and the endosperm. The endosperm plays a major role in the regulation of seed size. In Arabidopsis thaliana, endosperm size depends on the coordination of the genetic pathway HAIKU (IKU) with epigenetic controls comprising genome dosage, DNA methylation, and trimethylated lysine 27 on histone H3 (H3K27me3) deposition. However, the effectors that integrate these pathways have remained unknown. Here, we identify a target of the IKU pathway, the cytokinin oxidase CKX2, that affects cytokinin signaling. CKX2 expression is activated by the IKU transcription factor WRKY10 directly and promotes endosperm growth. CKX2 expression also depends on H3K27me3 deposition, which fluctuates in response to maternal genome dosage imbalance and DNA demethylation of male gametes. Hence, the control of endosperm growth by CKX2 integrates genetic and epigenetic regulations. In angiosperms, cytokinins are highly active in endosperm, and we propose that IKU effectors coordinate environmental and physiological factors, resulting in modulation of seed size.

Enhancing sucrose synthase activity results in increased levels of starch and ADP-glucose in maize (Zea mays L.) seed endosperms.

Abstract: Sucrose synthase (SuSy) is a highly regulated cytosolic enzyme that catalyzes the conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate glucose and fructose. In cereal endosperms, it is widely assumed that the stepwise reactions of SuSy, UDPglucose pyrophosphorylase and ADPglucose (ADPG) pyrophosphorylase (AGP) take place in the cytosol to convert sucrose into ADPG necessary for starch biosynthesis, although it has also been suggested that SuSy may participate in the direct conversion of sucrose into ADPG. In this study, the levels of the major primary carbon metabolites, and the activities of starch metabolism-related enzymes were assessed in endosperms of transgenic maize plants ectopically expressing StSUS4, which encodes a potato SuSy isoform. A total of 29 fertile lines transformed with StSUS4 were obtained, five of them containing a single copy of the transgene that was still functional after five generations. The number of seeds per ear of the five transgenic lines containing a single StSUS4 copy was comparable with that of wild-type (WT) control seeds. However, transgenic seeds accumulated 10-15% more starch at the mature stage, and contained a higher amylose/amylopectin balance than WT seeds. Endosperms of developing StSUS4-expressing seeds exhibited a significant increase in SuSy activity, and in starch and ADPG contents when compared with WT endosperms. No significant changes could be detected in the transgenic seeds in the content of soluble sugars, and in activities of starch metabolism-related enzymes when compared with WT seeds. A suggested metabolic model is presented wherein both AGP and SuSy are involved in the production of ADPG linked to starch biosynthesis in maize endosperm cells.

Iron biofortification of rice using different transgenic approaches

Abstract: More than 2 billion people suffer from iron (Fe) deficiency, and developing crop cultivars with an increased concentration of micronutrients (biofortification) can address this problem. In this review, we describe seven transgenic approaches, and combinations thereof, that can be used to increase the concentration of Fe in rice seeds. The first approach is to enhance the Fe storage capacity of grains through expression of the Fe storage protein ferritin under the control of endosperm-specific promoters. Using this approach, the concentration of Fe in the seeds of transformants was increased by approximately 2-fold in polished seeds. The second approach is to enhance Fe translocation by overproducing the natural metal chelator nicotianamine; using this approach, the Fe concentration was increased by up to 3-fold in polished seeds. The third approach is to enhance Fe influx to the endosperm by expressing the Fe(II)-nicotianamine transporter gene OsYSL2 under the control of an endosperm-specific promoter and sucrose transporter promoter, which increased the Fe concentration by up to 4-fold in polished seeds. The fourth approach is introduction of the barley mugineic acid synthesis gene IDS3 to enhance Fe uptake and translocation within plants, which resulted in a 1.4-fold increase in the Fe concentration in polished seeds during field cultivation. In addition to the above approaches, Fe-biofortified rice was produced using a combination of the first, second, and third approaches. The Fe concentration in greenhouse-grown T2 polished seeds was 6-fold higher and that in paddy field-grown T3 polished seeds was 4.4-fold higher than in non-transgenic seeds without any reduction in yield. When the first and fourth approaches were combined, the Fe concentration was greater than that achieved by introducing only the ferritin gene, and Fe-deficiency tolerance was observed. With respect to Fe biofortification, the introduction of multiple Fe homeostasis genes is more effective than the introduction of individual genes. Moreover, three additional approaches, i.e., overexpression of the Fe transporter gene OsIRT1 or OsYSL15, overexpression of the Fe deficiency-inducible bHLH transcription factor OsIRO2, and knockdown of the vacuolar Fe transporter gene OsVIT1 or OsVIT2, may be useful to further increase the Fe concentration of seeds.

Dynamic Expression of Imprinted Genes Associates with Maternally Controlled Nutrient Allocation during Maize Endosperm Development

Abstract: In angiosperms, the endosperm provides nutrients for embryogenesis and seed germination and is the primary tissue where gene imprinting occurs. To identify the imprintome of early developing maize (Zea mays) endosperm, we performed high-throughput transcriptome sequencing of whole kernels at 0, 3, and 5 d after pollination (DAP) and endosperms at 7, 10, and 15 DAP, using B73 by Mo17 reciprocal crosses. We observed gradually increased expression of paternal transcripts in 3- and 5-DAP kernels. In 7-DAP endosperm, the majority of the genes tested reached a 2:1 maternal versus paternal ratio, suggesting that paternal genes are nearly fully activated by 7 DAP. A total of 116, 234, and 63 genes exhibiting parent-specific expression were identified at 7, 10, and 15 DAP, respectively. The largest proportion of paternally expressed genes was at 7 DAP, mainly due to the significantly deviated parental allele expression ratio of these genes at this stage, while nearly 80% of the maternally expressed genes (MEGs) were specific to 10 DAP and were primarily attributed to sharply increased expression levels compared with the other stages. Gene ontology enrichment analysis of the imprinted genes suggested that 10-DAP endosperm-specific MEGs are involved in nutrient uptake and allocation and the auxin signaling pathway, coincident with the onset of starch and storage protein accumulation.

Polycomb Group Gene OsFIE2 Regulates Rice (Oryza sativa) Seed Development and Grain Filling via a Mechanism Distinct from Arabidopsis

Abstract: Cereal endosperm represents 60% of the calories consumed by human beings worldwide. In addition, cereals also serve as the primary feedstock for livestock. However, the regulatory mechanism of cereal endosperm and seed development is largely unknown. Polycomb complex has been shown to play a key role in the regulation of endosperm development in Arabidopsis, but its role in cereal endosperm development remains obscure. Additionally, the enzyme activities of the polycomb complexes have not been demonstrated in plants. Here we purified the rice OsFIE2-polycomb complex using tandem affinity purification and demonstrated its specific H3 methyltransferase activity. We found that the OsFIE2 gene product was responsible for H3K27me3 production specifically in vivo. Genetic studies showed that a reduction of OsFIE2 expression led to smaller seeds, partially filled seeds, and partial loss of seed dormancy. Gene expression and proteomics analyses found that the starch synthesis rate limiting step enzyme and multiple storage proteins are down-regulated in OsFIE2 reduction lines. Genome wide ChIP–Seq data analysis shows that H3K27me3 is associated with many genes in the young seeds. The H3K27me3 modification and gene expression in a key helix-loop-helix transcription factor is shown to be regulated by OsFIE2. Our results suggest that OsFIE2-polycomb complex positively regulates rice endosperm development and grain filling via a mechanism highly different from that in Arabidopsis.

ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2

Abstract: Seed production in angiosperms requires tight coordination of the development of the embryo and the endosperm. The endosperm-specific transcription factor ZHOUPI has previously been shown to play a key role in this process, by regulating both endosperm breakdown and the formation of the embryonic cuticle. To what extent these processes are functionally linked is, however, unclear. In order to address this issue we have concentrated on the subtilisin-like serine protease encoding gene ABNORMAL LEAF-SHAPE1. Expression of ABNORMAL LEAF-SHAPE1 is endosperm specific, and dramatically decreased in zhoupi mutants. We show that, although ABNORMAL LEAF-SHAPE1 is required for normal embryonic cuticle formation, it plays no role in regulating endosperm breakdown. Furthermore, we show that re-introducing ABNORMAL LEAF-SHAPE1 expression in the endosperm of zhoupi mutants partially rescues embryonic cuticle formation without rescuing their persistent endosperm phenotype. Thus, we conclude that ALE1 can normalize cuticle formation in the absence of endosperm breakdown, and that ZHOUPI thus controls two genetically separable developmental processes. Finally, our genetic study shows that ZHOUPI and ABNORMAL LEAF-SHAPE1 promotes formation of embryonic cuticle via a pathway involving embryonically expressed receptor kinases GASSHO1 and GASSHO2. We therefore provide a molecular framework of inter-tissue communication for embryo-specific cuticle formation during embryogenesis.

Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm

Abstract: The endosperm of cereal grains is one of the most valuable products of modern agriculture. Cereal endosperm development comprises different phases characterized by mitotic cell proliferation, endoreduplication, the accumulation of storage compounds, and programmed cell death. Although manipulation of these processes could maximize grain yield, how they are regulated and integrated is poorly understood. We show that the Retinoblastoma-related (RBR) pathway controls key aspects of endosperm development in maize. Down-regulation of RBR1 by RNAi resulted in up-regulation of RBR3-type genes, as well as the MINICHROMOSOME MAINTENANCE 2-7 gene family and PROLIFERATING CELL NUCLEAR ANTIGEN, which encode essential DNA replication factors. Both the mitotic and endoreduplication cell cycles were stimulated. Developing transgenic endosperm contained 42-58% more cells and ∼70% more DNA than wild type, whereas there was a reduction in cell and nuclear sizes. In addition, cell death was enhanced. The DNA content of mature endosperm increased 43% upon RBR1 down-regulation, whereas storage protein content and kernel weight were essentially not affected. Down-regulation of both RBR1 and CYCLIN DEPENDENT KINASE A (CDKA);1 indicated that CDKA;1 is epistatic to RBR1 and controls endoreduplication through an RBR1-dependent pathway. However, the repressive activity of RBR1 on downstream targets was independent from CDKA;1, suggesting diversification of RBR1 activities. Furthermore, RBR1 negatively regulated CDK activity, suggesting the presence of a feedback loop. These results indicate that the RBR1 pathway plays a major role in regulation of different processes during maize endosperm development and suggest the presence of tissue/organ-level regulation of endosperm/seed homeostasis.

Kin conflict in seed development: an interdependent but fractious collective.

Abstract: Seeds are complex structures that unite diploid maternal tissues with filial tissues that may be haploid (gametophyte), diploid (embryo), or triploid (endosperm). Maternal tissues are predicted to favor smaller seeds than are favored by filial tissues, and filial genes of maternal origin are predicted to favor smaller seeds than are favored by filial genes of paternal origin. Consistent with these predictions, seed size is determined by an interplay between growth of maternal integuments, which limits seed size, and of filial endosperm, which promotes larger seeds. Within endosperm, genes of paternal origin favor delayed cellularization of endosperm and larger seeds, whereas genes of maternal origin favor early cellularization and smaller seeds. The ratio of maternal and paternal gene products in endosperm contributes to the failure of crosses between different ploidy levels of the same species and crosses between species. Maternally expressed small-interfering RNAs (siRNAs) are predicted to associate with growth-enhancing genes.

Comparative proteomic analysis of the effect of temperature and fertilizer on gliadin and glutenin accumulation in the developing endosperm and flour from Triticum aestivum L. cv. Butte 86

Abstract: Flour quality is largely determined by the gluten proteins, a complex mixture of proteins consisting of high molecular weight-glutenin subunits (HMW-GS), low molecular weight-glutenin subunits (LMW-GS), and α-, γ-, and ω-gliadins. Detailed proteomic analyses of the effects of fertilizer and high temperature on individual gliadin and glutenin protein levels are needed to determine how these environmental factors influence flour quality. Wheat plants (Triticum aestivum L. cv. Butte 86) were grown in greenhouses under moderate and high temperature regimens with and without post-anthesis fertilizer. Quantitative two-dimensional gel electrophoresis was used to construct accumulation profiles in developing endosperm for the entire complement of gluten proteins identified previously by tandem mass spectrometry. Amounts of individual gliadins and glutenins were also determined in flour produced under each of the regimens. Under all environmental regimens, most HMW-GS, LMW-GS, γ- and ω-gliadins accumulated rapidly during early stages of grain development and leveled off during middle stages of development. A subset of LMW-GS showed a second distinct profile, accumulating throughout development, while α-gliadins showed a variety of accumulation profiles. In flour, fourteen distinct gluten proteins responded similarly to fertilizer, high temperature, and high temperature plus fertilizer. The majority of HMW-GS and ω-gliadins and some α-gliadins increased while two LMW-GS and a minor γ-gliadin decreased. Fertilizer did not influence gluten protein accumulation under high temperature conditions. Additionally, the effects of fertilizer and high temperature were not additive; very few changes were observed when plants that received fertilizer were subjected to high temperature. Although post-anthesis temperature and fertilizer have very different effects on grain development and yield, the two treatments elicit surprisingly similar effects on the accumulation of gluten proteins. The similarity of the responses to the different treatments is likely due to source-sink activities of nitrogen reserves in the wheat plant. Because each protein that showed a response in this study is linked to a gene sequence, the work sets the stage for transgenic studies that will better elucidate the roles of specific proteins in flour quality and in the response to the environment.

Functional analyses of TaHMA2, a P(1B)-type ATPase in wheat.

Abstract: Currently, there are few studies concerning the function of heavy metal ATPase 2 (HMA2), particularly in monocotyledons, and the potential application of this protein in biofortification and phytoremediation. Thus, we isolated and characterized the TaHMA2 gene from wheat (Triticum aestivum L.). Our results indicate that TaHMA2 is localized to the plasma membrane and stably expressed, except in the nodes, which showed relatively high expression. Zinc/cadmium (Zn/Cd) resistance was observed in TaHMA2-transformed yeast. The over-expression of TaHMA2 increased the elongation and decreased the seed-setting rate in rice (Oryza sativa L.), but not Arabidopsis thaliana, tobacco (Nicotiana tabacum L.) or wheat. TaHMA2 over-expression also improved root-shoot Zn/Cd translocation, especially in rice. The seeds of transgenic rice and wheat, not tobacco, showed decreased Zn concentrations. The Zn concentration was decreased in all parts of the transgenic rice seeds, but was decreased only in the ventral endosperm of wheat, which showed an increased Zn concentration in the embryo and aleurone. The over-expression of TaHMA2 improved plant tolerance under moderate Zn stress and Zn deficiency, but Zn and Cd resistance decreased under high levels of Zn and Cd stress, respectively. The Cd concentration in transgenic rice seedlings was dramatically increased under Zn deficiency. Thus, over-expression of TaHMA2 showed a more obvious phenotype in monocotyledons than in dicotyledons. These findings provide important information for TaHMA2, and more efforts should be made in the future to characterize the reduced Zn concentration in TaHMA2 transgenic grains and the diversity of TaHMA2 substrate specificity.

The differential transcription network between embryo and endosperm in the early developing maize seed

Abstract: Transcriptome analysis of early-developing maize (Zea mays) seed was conducted using Illumina sequencing. We mapped 11,074,508 and 11,495,788 paired-end reads from endosperm and embryo, respectively, at 9 d after pollination to define gene structure and alternative splicing events as well as transcriptional regulators of gene expression to quantify transcript abundance in both embryo and endosperm. We identified a large number of novel transcribed regions that did not fall within maize annotated regions, and many of the novel transcribed regions were tissue-specifically expressed. We found that 50.7% (8,556 of 16,878) of multiexonic genes were alternatively spliced, and some transcript isoforms were specifically expressed either in endosperm or in embryo. In addition, a total of 46 trans-splicing events, with nine intrachromosomal events and 37 interchromosomal events, were found in our data set. Many metabolic activities were specifically assigned to endosperm and embryo, such as starch biosynthesis in endosperm and lipid biosynthesis in embryo. Finally, a number of transcription factors and imprinting genes were found to be specifically expressed in embryo or endosperm. This data set will aid in understanding how embryo/endosperm development in maize is differentially regulated.

Phytochemicals and Antioxidant Capacity from Nypa fruticans Wurmb. Fruit.

Abstract: Nypa fruticans Wurmb. is one of the important underutilized fruit of Malaysia, which lacks scientific attention. Total phenolics, flavonoid content, and antioxidant capacities from endosperm extracts of Nypa fruticans (unripe and ripe fruits) were evaluated. Endosperm extract of unripe fruits (EEU) exhibited the highest phenolics ( mg GAE/g), flavonoid content ( RE/g), and antioxidant capacity. Free radical scavenging capacity of EEU as assessed by 2--azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picryl hydrazyl (DPPH) radicals showed inhibitory activity of % and %, respectively. Beta carotene bleaching coefficient of EEU was higher (), when compared to endosperm extract of ripe fruits (). Additionally, EEU exhibited high antioxidant capacity by phosphomolybdenum method and ferric reducing antioxidant power values. Eight phenolic compounds from Nypa fruticans endosperm extracts were identified and quantified by ultra-high-performance liquid chromatography. Chlorogenic acid, protocatechuic acid, and kaempferol were the major phenolic compounds. Thus this fruit could be used as a potential source of natural antioxidant.

Identification and characterization of lysine-rich proteins and starch biosynthesis genes in the opaque2 mutant by transcriptional and proteomic analysis

Abstract: Background The opaque2 mutant is valuable for producing maize varieties with enhanced nutritional value. However, the exact mechanisms by which it improves protein quality and creates a soft endosperm texture are unclear. Given the importance of improving nutritional quality in grain crops, a better understanding of the physiological basis for these traits is necessary.

RNA Interference Suppression of Genes in Glycosyl Transferase Families 43 and 47 in Wheat Starchy Endosperm Causes Large Decreases in Arabinoxylan Content

Abstract: The cell walls of wheat (Triticum aestivum) starchy endosperm are dominated by arabinoxylan (AX), accounting for 65% to 70% of the polysaccharide content. Genes within two glycosyl transferase (GT) families, GT43 (IRREGULAR XYLEM9 [IRX9] and IRX14) and GT47 (IRX10), have previously been shown to be involved in the synthesis of the xylan backbone in Arabidopsis, and close homologs of these have been implicated in the synthesis of xylan in other species. Here, homologs of IRX10 TaGT47_2 and IRX9 TaGT43_2, which are highly expressed in wheat starchy endosperm cells, were suppressed by RNA interference (RNAi) constructs driven by a starchy endosperm-specific promoter. The total amount of AX was decreased by 40% to 50% and the degree of arabinosylation was increased by 25% to 30% in transgenic lines carrying either of the transgenes. The cell walls of starchy endosperm in sections of grain from TaGT43_2 and TaGT47_2 RNAi transgenics showed decreased immunolabeling for xylan and arabinoxylan epitopes and approximately 50% decreased cell wall thickness compared with controls. The proportion of AX that was water soluble was not significantly affected, but average AX polymer chain length was decreased in both TaGT43_2 and TaGT47_2 RNAi transgenics. However, the long AX chains seen in controls were absent in TaGT43_2 RNAi transgenics but still present in TaGT47_2 RNAi transgenics. The results support an emerging picture of IRX9-like and IRX10-like proteins acting as key components in the xylan synthesis machinery in both dicots and grasses. Since AX is the main component of dietary fiber in wheat foods, the TaGT43_2 and TaGT47_2 genes are of major importance to human nutrition.