Development and function of caryopsis transport tissues in maize,sorghum and wheat

Cereal caryopsis transport tissues are essential channels via which nutrients are transported into the embryo and endosperm. There are differences and similarities between caryopsis transport tissues of maize, sorghum and wheat. Vascular bundle, endosperm transfer cells, endosperm conducting cells and embryo surrounding region are common in maize, sorghum and wheat. Placentochalaza is special in maize and sorghum, while chalaza and nucellar projection transfer cells are special in wheat. There is an obvious apoplastic cavity between maternal and filial tissues in sorghum and wheat caryopses, but there is no obvious apoplastic cavity in maize caryopsis. Based on the latest research, the development and function of the three cereal caryopsis transport tissues are discussed and investigated in this paper.     

Rice caryopsis development I: Dynamic changes in different cell layers

Rice caryopsis as one of the most important food sources for humans has a complex structure that is composed of maternal tissues including the pericarp and testa and filial tissues including the endosperm and embryo. Although rice caryopsis studies have been conducted previously, a systematic characterization throughout the entire developmental process is still lacking. In this study, detailed morphological examinations of caryopses were made during the entire 30‐day developmental process. We observed some rapid changes in cell differentiation events and cataloged how cellular degeneration processes occurred in maternal tissues. The differentiations of tube cells and cross cells were achieved by 9 days after pollination (DAP). In the testa, the outer integument was degenerated by 3 DAP, while the outer layer of the inner integument degenerated by 7 DAP. In the nucellus, all tissues with the exception of the nucellar projection and the nucellar epidermis degenerated in the first 5 DAP. By 21 DAP, all maternal tissues, including vascular bundles, the nucellar projection and the nucellar epidermal cells were degenerated. In summary, this study provides a complete atlas of the dynamic changes in cell differentiation and degeneration for individual maternal cell layers of rice caryopsis.     

Rice caryopsis development Ⅰ: Dynamic changes in different cell layers

Rice caryopsis as one of the most important food sources for humans has a complex structure that is composed of maternal tissues including the pericarp and testa and filial tissues including the endosperm and embryo. Although rice caryopsis studies have been conducted previously, a systematic characterization throughout the entire developmental process is still lacking. In this study, detailed morphological examinations of caryopses were made during the entire 30-day developmental process. We observed some rapid changes in cell differentiation events and cataloged how cellular degeneration processes occurred in maternal tissues. The differentiations of tube cells and cross cells were achieved by 9 days after pollination(DAP). In the testa, the outer integument was degenerated by 3 DAP, while the outer layer of the inner integument degenerated by 7 DAP. In the nucellus, all tissues with the exception of the nucellar projection and the nucellar epidermis degenerated in the first 5 DAP. By 21 DAP, all maternal tissues, including vascular bundles, the nucellar projection and the nucellar epidermal cells were degenerated. In summary, this study provides a complete atlas of the dynamic changes in cell differentiation and degeneration for individual maternal cell layers of rice caryopsis.     

Development of maternal seed tissue in barley is mediated by regulated cell expansion and cell disintegration and coordinated with endosperm growth

After fertilization, filial grain organs are surrounded by the maternal nucellus embedded within the integuments and pericarp. Rapid early endosperm growth must be coordinated with maternal tissue development. Parameters of maternal tissue growth and development were analysed during early endosperm formation. In the pericarp, cell proliferation is accomplished around the time of fertilization, followed by cell elongation predominantly in longitudinal directions. The rapid cell expansion coincides with endosperm cellularization. Distribution of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei reveals distinct patterns starting in the nucellus at anthesis and followed later by the inner cell rows of the pericarp, then spreading to the whole pericarp. The pattern suggests timely and spatially regulated programmed cell death (PCD) processes in maternal seed tissues. When the endosperm is coenocytic, PCD events are only observed within the nucellus. Thereby, remobilization of nucellar storage compounds by PCD could nourish the early developing endosperm when functional interconnections are absent between maternal and filial seed organs. Specific proteases promote PCD events. Characterization of the barley vacuolar processing enzyme (VPE) gene family identified seven gene members specifically expressed in the developing grain. HvVPE2a (known as nucellain) together with closely similar HvVPE2b and HvVPE2d might be involved in nucellar PCD. HvVPE4 is strongly cell specific for pericarp parenchyma. Correlative evidence suggests that HvVPE4 plays a role in PCD events in the pericarp. Possible functions of PCD in the maternal tissues imply a potential nutritive role or the relief of a physical restraint for endosperm growth. PCD could also activate post-phloem transport functions.     

Maternal control of nutrient allocation in plant seeds by genomic imprinting

Imprinted genes are commonly expressed in mammalian placentas and in plant seed endosperms, where they exhibit preferential uniparental allelic expression. In mammals, imprinted genes directly regulate placental function and nutrient distribution from mother to fetus; however, none of the >60 imprinted genes thus far reported in plants have been demonstrated to play an equivalent role in regulating the flow of resources to the embryo. Here we show that imprinted Maternally expressed gene1 (Meg1) in maize is both necessary and sufficient for the establishment and differentiation of the endosperm nutrient transfer cells located at the mother:seed interface. Consistent with these findings, Meg1 also regulates maternal nutrient uptake, sucrose partitioning, and seed biomass yield. In addition, we generated an imprinted and nonimprinted synthetic Meg1 ((syn)Meg1) dosage series whereby increased dosage and absence of imprinting both resulted in an unequal investment of maternal resources into the endosperm. These findings highlight dosage regulation by genomic imprinting as being critical for maintaining a balanced distribution of maternal nutrients to filial tissues in plants, as in mammals. However, unlike in mammals, Meg1 is a maternally expressed imprinted gene that surprisingly acts to promote rather than restrict nutrient allocation to the offspring.     

Live and Let Die - The Bsister MADS-Box Gene OsMADS29 Controls the Degeneration of Cells in Maternal Tissues during Seed Development of Rice (Oryza sativa)

B_sister genes have been identified as the closest relatives of class B floral homeotic genes. Previous studies have shown that B_sister genes from eudicots are involved in cell differentiation during ovule and seed development. However, the complete function of B_sister genes in eudicots is masked by redundancy with other genes and little is known about the function of B_sister genes in monocots, and about the evolution of B_sister gene functions. Here we characterize OsMADS29, one of three MADS-box B_sister genes in rice. Our analyses show that OsMADS29 is expressed in female reproductive organs including the ovule, ovule vasculature, and the whole seed except for the outer layer cells of the pericarp. Knock-down of OsMADS29 by double-stranded RNA-mediated interference (RNAi) results in shriveled and/or aborted seeds. Histological analyses of the abnormal seeds at 7 days after pollination (DAP) indicate that the symplastic continuity, including the ovular vascular trace and the nucellar projection, which is the nutrient source for the filial tissue at early development stages, is affected. Moreover, degeneration of all the maternal tissues in the transgenic seeds, including the pericarp, ovular vascular trace, integuments, nucellar epidermis and nucellar projection, is blocked as compared to control plants. Our results suggest that OsMADS29 has important functions in seed development of rice by regulating cell degeneration of maternal tissues. Our findings provide important insights into the ancestral function of B_sister genes.     

Sucrose transport into barley seeds: molecular characterization of two transporters and implications for seed development and starch accumulation

In order to understand sucrose transport in developing seeds of cereals at the molecular level, we cloned from a caryopses library two cDNAs encoding sucrose transporters, designated HvSUT1 and HvSUT2. Sucrose uptake activity was confirmed by heterologous expression in yeast. Both transporter genes are expressed in maternal as well as filial tissues. In a series of in situ hybridizations we analysed the cell type-specific expression in developing seeds. HvSUT1 is preferentially expressed in caryopses in the cells of the nucellar projection and the endospermal transfer layer, which represent the sites of sucrose exchange between the maternal and the filial generation and are characterized by transfer cell formation. HvSUT2 is expressed in all sink and source tissues analysed and may have a general housekeeping role. The rapid induction of HvSUT1 gene expression in caryopses at approximately 5-6 days after fertilization coincides with increasing levels of sucrose as well as sucrose synthase mRNA and activity, and occurs immediately before the onset of rapid starch accumulation within the endosperm. Starch biosynthesis requires sucrose to be imported into the endosperm, as direct precursor for starch synthesis and to promote storage-associated processes. We discuss the possible role of HvSUT1 as a control element for the endospermal sucrose concentration.     

The promoter of the asi gene directs expression in the maternal tissues of the seed in transgenic barley

The bifunctional alpha-amylase/subtilisin inhibitor (BASI) is an abundant protein in barley seeds, proposed to play multiple and apparently diverse roles in regulation of starch hydrolysis and in seed defence against pathogens. In the Triticeae, the protein has evolved the ability to specifically inhibit the main group of alpha-amylases expressed during germination of barley and encoded by the amyl gene family found only in the Triticeae. The expression of the asi gene that encodes BASI has been reported to be controlled by the hormones abscisic acid (ABA) and gibberellic acid (GA). Despite many studies at the gene and protein level, the function of this gene in the plant remains unclear. In this study, the 5'-flanking region (1033 bp, 1033-asi promoter) and the 3'-flanking region (655 bp) of the asi gene were isolated and characterised. The 1033-asi promoter sequence showed homology to a number of ciselements that play a role in ABA and GA regulated expression of other genes. With a green fluorescent protein gene (gfp) as reporter, the 1033-asi promoter was studied for spatial, temporal and hormonal control of gene expression. The 1033-asi promoter and its deletions direct transient gfp expression in the pericarp and at low levels in mature aleurone cells, and this expression is not regulated by ABA or GA. In transgenic barley plants, the 1033-asi promoter directed tissue-specific expression of the gfp gene in developing grain and germinating grain but not in roots or leaves. In developing grain, expression of gfp was observed specifically in the pericarp, the vascular tissue, the nucellar projection cells and the endosperm transfer cells and the hormones ABA or GA did not regulate this expression. In mature germinating grain gfp expression was observed in the embryo but not in aleurone or starchy endosperm. However, GA induced gfp expression in the aleurone of mature imbibed seeds from which the embryo had been removed. Expression in maternal rather than endosperm tissues of the grain suggests that earlier widespread assumptions that the protein is expressed largely in the endosperm may have been largely based on analysis of mixed grain tissues. This novel pattern of expression suggests that both activities of the protein may be primarily involved in seed defence in the peripheral tissues of the seed.     

Spatial and temporal profiles of cytokinin biosynthesis and accumulation in developing caryopses of maize

Background and Aims

Cytokinins are a major group of plant hormones and are associated with various developmental processes. Developing caryopses of maize have high levels of cytokinins, but little is known about their spatial and temporal distribution. The localization and quantification of cytokinins was investigated in maize (Zea mays) caryopsis from 0 to 28 d after pollination together with the expression and localization of isopentenyltransferase ZmIPT1 involved in cytokinin biosynthesis and ZmCNGT, the gene putatively involved in N9-glucosylation.

Methods

Biochemical, cellular and molecular approaches resolved the overall cytokinin profiles, and several gene expression assays were used for two critical genes to assess cytokinin cell-specific biosynthesis and conversion to the biologically inactive form. Cytokinins were immunolocalized for the first time in maize caryopses.

Key Results

During the period 0–28 d after pollination (DAP): (1) large quantities of cytokinins were detected in the maternal pedicel region relative to the filial tissues during the early stages after fertilization; (2) unpollinated ovules did not accumulate cytokinins; (3) the maternal nucellar region showed little or no cytokinin signal; (4) the highest cytokinin concentrations in filial endosperm and embryo were detected at 12 DAP, predominantly zeatin riboside and zeatin-9-glucoside, respectively; and (5) a strong cytokinin immuno-signal was detected in specific cell types in the pedicel, endosperm and embryo.

Conclusions

The cytokinins of developing maize caryopsis may originate from both local syntheses as well as by transport. High levels of fertilization-dependent cytokinins in the pedicel suggest filial control on metabolism in the maternal tissue; they may also trigger developmental programmed cell death in the pedicel.     

Water uptake and splitting of wild oat caryopsis

Imbibition and germination experiments were conducted on the caryopses of wild oats (Avena fatua L.). The embryo envelopes, pericarp and aleurone layer, which completely cover the embryo-endosperm, do not form barriers against water uptake. The initial uptake of water is passive and the water moves across the pericarp with ease as it contains cracks; it is, however, transported across the aleurone layer through its cell walls into the endosperm and embryo of the caryopsis. The starchy endosperm enlarges due to water uptake causing the pericarp to rupture, thus exposing the aleuronelayer-covered seed. The aleurone layer is structurally heterogenous consistings of radially compressed irregular cells and cuboidal or radiallys tretched cells; the latter contains thicker walls. The former type is present along the abaxial side of the embryo and in the crease on the adaxial side of the caryopsis; the latter type covers the endosperm. The physical distention of the endosperm due to water uptake causes the rupture of the pericarp and the aleurone layer, and facilitates the emergence of the radicle and coleorhiza of the embryo during caryopsis germination.     

Systematic spatial analysis of gene expression during wheat caryopsis development

The cereal caryopsis is a complex tissue in which maternal and endosperm tissues follow distinct but coordinated developmental programs. Because of the hexaploid genome in wheat (Triticum aestivum), the identification of genes involved in key developmental processes by genetic approaches has been difficult. To bypass this limitation, we surveyed 888 genes that are expressed during caryopsis development using a novel high-throughput mRNA in situ hybridization method. This survey revealed novel distinct spatial expression patterns that either reflected the ontogeny of the developing caryopsis or indicated specialized cellular functions. We have identified both known and novel genes whose expression is cell cycle-dependent. We have identified the crease region as important in setting up the developmental patterning, because the transition from proliferation to differentiation spreads from this region to the rest of the endosperm. A comparison of this set of genes with the rice (Oryza sativa) genome shows that approximately two-thirds have rice counterparts but also suggests considerable divergence with regard to proteins involved in grain filling. We found that the wheat genes had significant homology with 350 Arabidopsis thaliana genes. At least 25 of these are already known to be essential for seed development in Arabidopsis, but many others remain to be characterized.     

Caspase-Like Activities Accompany Programmed Cell Death Events in Developing Barley Grains

Programmed cell death is essential part of development and cell homeostasis of any multicellular organism. We have analyzed programmed cell death in developing barley caryopsis at histological, biochemical and molecular level. Caspase-1, -3, -4, -6 and -8-like activities increased with aging of pericarp coinciding with abundance of TUNEL positive nuclei and expression of HvVPE4 and HvPhS2 genes in the tissue. TUNEL-positive nuclei were also detected in nucellus and nucellar projection as well as in embryo surrounding region during early caryopsis development. Quantitative RT-PCR analysis of micro-dissected grain tissues revealed the expression of HvVPE2a, HvVPE2b, HvVPE2d, HvPhS2 and HvPhS3 genes exclusively in the nucellus/nucellar projection. The first increase in cascade of caspase-1, -3, -4, -6 and -8-like activities in the endosperm fraction may be related to programmed cell death in the nucellus and nucellar projection. The second increase of all above caspase-like activities including of caspase-9-like was detected in the maturating endosperm and coincided with expression of HvVPE1 and HvPhS1 genes as well as with degeneration of nuclei in starchy endosperm and transfer cells. The distribution of the TUNEL-positive nuclei, tissues-specific expression of genes encoding proteases with potential caspase activities and cascades of caspase-like activities suggest that each seed tissue follows individual pattern of development and disintegration, which however harmonizes with growth of the other tissues in order to achieve proper caryopsis development.