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Germination of the coffee (Coffea arabica L.) seed is the result of events that occur simultaneously in the embryo and endosperm. To understand the molecular mechanisms responsible for these events, we undertook a transcriptome analysis of embryo, micropylar and lateral endosperms from 10-day-imbibed seeds. The sequencing yielded contigs coding for 16,813 proteins. From those, 14,005 (~ 83%) were highly similar to at least one protein sequence in the nr database. 162 genes were significantly expressed in the embryo, 36 in the micropylar endosperm and 72 in the lateral endosperm. The tissue specificity analysis of the significantly expressed genes showed that the embryo had the highest proportion of specific genes (113/162, ~70%), while 11 were expressed in the micropylar and lateral endosperms. In the embryo, genes were mainly associated with abiotic stress, cell growth, and intercellular communication. In the micropylar and lateral endosperms, they were associated with abiotic stress and cell wall degradation. The accuracy of RNA-seq data was confirmed by RT-qPCR. This work adds new information about the molecular mechanism involved in coffee seed germination. Resumo: A germinação da semente do café (Coffea arabica L.) é resultado de eventos que ocorrem simultaneamente no embrião e endosperma. Para entender os mecanismos moleculares responsáveis por estes eventos, nós realizamos uma análise transcricional do embrião e dos endospermas micropilar e lateral de sementes embebidas por 10 dias. O sequenciamento obteve contigs que codificam 16.813 proteínas. Destas, 14.005 (~ 83%) foram altamente similares a pelo menos uma sequência de proteína na base de dados nr. 162 genes foram significativamente expressos em embrião, 36 em endosperma micropilar e 72 em endosperma lateral. A análise de tecido-especificidade dos genes significativamente expressos mostrou que a maior proporção de genes específicos está no embrião (113/162, ~70%), enquanto 11 genes foram expressos nos endospermas lateral e micropilar. No embrião, os genes foram associados principalmente com estresse abiótico, crescimento celular e comunicação intercelular. Nos endospermas micropilar e lateral, eles foram associados com estresse abiótico e degradação da parede celular. A acurácia da análise de RNA-seq foi confirmada por RT-qPCR. Este trabalho acrescenta novas informações sobre os mecanismos moleculares envolvidos na germinação de sementes de café.

In this study, a non-destructive, high-throughput, endosperm-based DNA extraction method was developed. To verify the non-destructive nature of this method, a germination test was performed on 288 seeds after sampling their endosperm, which gave a seedling emergence rate that was higher (97.6%) than that of the control group (92%). To confirm the feasibility of the new method, DNA was extracted from plants of a BC1F2 population by two different methods, namely, from endosperm using our rapid, high-throughput method (ER-DNA) and from young leaves emerging from the same sampled seed using the CTAB method (LC-DNA). The ER-DNA was undetectable by agarose gel electrophoresis, but was found to be an adequate replacement for LC-DNA for the amplification and detection of simple sequence repeats (SSRs). Further analysis revealed that ER-DNA was generally suitable for the generation of specific 500-750-bp fragments, but not for the amplification of 1,000-2,000-bp fragments. Our rapid, high-throughput method therefore has no deleterious effects on wheat seeds and yields DNA for SSR genotyping that is a suitable alternative to traditionally obtained DNA. RESUMO: Neste estudo, foi desenvolvido um método de extração de DNA não destrutivo, de alto débito e endosperma. Para verificar a natureza não destrutiva deste método, um teste de germinação foi realizado em 288 sementes após a amostragem do endosperma, o que deu uma taxa de emergência de plântulas maior (97,6%) do que a do grupo controle (92%). Para confirmar a viabilidade do novo método, o DNA foi extraído de plantas de uma população de BC1F2 por dois métodos diferentes, a saber, do endosperma usando nosso método rápido de alto rendimento (ER-DNA) e de folhas jovens que emergem da mesma semente amostrada usando o método CTAB (LC-DNA). O ER-DNA foi indetectável por eletroforese em gel de agarose, mas foi encontrado como uma substituição adequada para LC-DNA para a amplificação e detecção de repetições simples de seqüência (SSRs). Uma análise posterior revelou que o ER-DNA era geralmente adequado para a geração de fragmentos específicos de 500-750 pb, mas não para a amplificação de fragmentos de 1.000-2.000 pb. Nosso método rápido e de alto débito, portanto, não tem efeitos deletérios sobre as sementes de trigo e produz DNA para a genotipagem de SSR que é uma alternativa adequada ao DNA obtido tradicionalmente.

▪ Abstract  Methylation of cytosine residues in DNA provides a mechanism of gene control. There are two classes of methyltransferase in Arabidopsis; one has a carboxy-terminal methyltransferase domain fused to an amino-terminal regulatory domain and is similar to mammalian methyltransferases. The second class apparently lacks an amino-terminal domain and is less well conserved. Methylcytosine can occur at any cytosine residue, but it is likely that clonal transmission of methylation patterns only occurs for cytosines in strand-symmetrical sequences CpG and CpNpG. In plants, as in mammals, DNA methylation has dual roles in defense against invading DNA and transposable elements and in gene regulation. Although originally reported as having no phenotypic consequence, reduced DNA methylation disrupts normal plant development.

The mechanisms that initiate reproductive development after fertilization are not understood. Reproduction in higher plants is unique because it is initiated by two fertilization events in the haploid female gametophyte. One sperm nucleus fertilizes the egg to form the embryo. A second sperm nucleus fertilizes the central cell to form the endosperm, a unique tissue that supports the growth of the embryo. Fertilization also activates maternal tissue differentiation, the ovule integuments form the seed coat, and the ovary forms the fruit. To investigate mechanisms that initiate reproductive development, a female-gametophytic mutation termed fie (fertilization-independent endosperm) has been isolated in Arabidopsis. The fie mutation specifically affects the central cell, allowing for replication of the central cell nucleus and endosperm development without fertilization. The fie mutation does not appear to affect the egg cell, suggesting that the processes that control the initiation of embryogenesis and endosperm development are different. FIE/fie seed coat and fruit undergo fertilization-independent differentiation, which shows that the fie female gametophyte is the source of signals that activates sporophytic fruit and seed coat development. The mutant fie allele is not transmitted by the female gametophyte. Inheritance of the mutant fie allele by the female gametophyte results in embryo abortion, even when the pollen bears the wild-type FIE allele. Thus, FIE carries out a novel, essential function for female reproductive development.

Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy. Within plastics, carbon can be used in the biosynthesis of starch or as a substrate for the oxidative pentose phosphate pathway, for example. We have used maize endosperm to purify a plastidic glucose 6-phosphate/phosphate translocator (GPT). The corresponding cDNA was isolated from maize endosperm as well as from tissues of pea roots and potato tubers. Analysis of the primary sequences of the cDNAs revealed that the GPT proteins have a high degree of identity with each other but share only approximately 38% identical amino acids with members of both the triose phosphate/phosphate translocator (TPT) and the phosphoenolpyruvate/phosphate translocator (PPT) families. Thus, the GPTs represent a third group of plastidic phosphate antiporters. All three classes of phosphate translocator genes show differential patterns of expression. Whereas the TPT gene is predominantly present in tissues that perform photosynthetic carbon metabolism and the PPT gene appears to be ubiquitously expressed, the expression of the GPT gene is mainly restricted to heterotrophic tissues. Expression of the coding region of the GPT in transformed yeast cells and subsequent transport experiments with the purified protein demonstrated that the GPT protein mediates a 1:1 exchange of glucose 6-phosphate mainly with inorganic phosphate and triose phosphates. Glucose 6-phosphate imported via the GPT can thus be used either for starch biosynthesis, during which process inorganic phosphate is released, or as a substrate for the oxidative pentose phosphate pathway, yielding triose phosphates

Homeobox genes are master regulatory genes that specify the body plan and control development of many eukaryotic organisms, including plants. We isolated and characterized a cDNA designated ATML1 (for Arabidopsis thaliana meristem L1 layer) that encodes a novel homeodomain protein. The ATML1 protein shares high sequence homology inside and outside of the homeodomain with both the Phalaenopsis 039 and the Arabidopsis GLABRA2 (GL2) homeodomain proteins, which together define a new class of plant homeodomain-containing proteins, designated HD-GL2. The ATML1 gene was first expressed in the apical cell after the first asymmetric division of the zygote and continued to be expressed in all proembryo cells until the eight-cell stage. In the 16-cell proembryo, the ATML1 gene showed a distinct pattern of expression, with its mRNA becoming restricted to the protoderm. In the torpedo stage of embryo development, ATML1 mRNA disappeared altogether but reappeared later only in the L1 layer of the shoot apical meristem in the mature embryo. After germination, this L1 layer-specific pattern of expression was maintained in the vegetative shoot apical meristem, inflorescence, and floral meristems, as well as in the young floral organ primordia. Finally, ATML1 mRNA accumulated in the protoderm of the ovule primordia and integuments and gradually became restricted in its expression to the endothelium surrounding the embryo sac. We propose that ATML1 may be involved in setting up morphogenetic boundaries of positional information necessary for controlling cell specification and pattern formation. In addition, ATML1 provides an early molecular marker for the establishment of both apical-basal and radial patterns during plant embryogenesis

Preparations enriched in plastics were used to investigate the location of ADP-glucose pyrophosphorylase (AGPase) in the developing endosperm of maize (Zea mays L.). These preparations contained more than 25% of the total activity of the plastic marker enzymes alkaline pyrophosphatase and soluble starch synthase, less than 2% of the cytosolic marker enzymes alcohol dehydrogenase and pyrophosphate, fructose 6-phosphate 1-phosphotransferase, and approximately 3% of the AGPase activity. Comparison with the marker enzyme distribution suggests that more than 95% of the activity of AGPase in maize endosperm is extra-plastidial. Two proteins were recognized by antibodies to the small subunit of AGPase from maize endosperm Brittle-2 (Bt2). The larger of the two proteins was the major small subunit in homogenates of maize endosperm, and the smaller, less abundant of the two proteins was enriched in preparations containing plastics. These results suggest that there are distinct plastidial and cytosolic forms of AGPase, which are composed of different subunits. Consistent with this was the finding that the bt2 mutation specifically eliminated the extra-plastidial AGPase activity and the larger of the two proteins recognized by the antibody to the Bt2 subunit

We characterized the progression of programmed cell death during maize (Zea mays L.) endosperm development of starchy (Su; wild-type) and shrunken2 (sh2) genotypes and tested the involvement of ethylene in mediating this process. Histological and viability staining demonstrated that endosperm cell death was initiated earlier and progressed more rapidly in sh2 endosperm compared with Su endosperm. Internucleosomal DNA fragmentation accompanied endosperm cell death and occurred more extensively in sh2 endosperm. 1-Aminocyclopropane-1-carboxylic acid levels peaked approximately 16 d after pollination (dap) in Su endosperm and gradually decreased during subsequent development, whereas two large 1-aminocyclopropane-1-carboxylic acid peaks were observed in sh2 endosperm, the first between 16 and 20 dap and the second at 36 dap. Ethylene levels were elevated in sh2 kernels compared with Su kernels, with an initial peak 20 dap approximately 3-fold higher than in Su kernels and a second peak 36 dap approximately 5-fold higher than that in Su kernels. Ethylene treatment of Su kernels resulted in earlier and more extensive endosperm cell death and DNA fragmentation. Aminoethoxyvinylglycine treatment of sh2 kernels reduced the extent of DNA fragmentation. We conclude that ethylene is involved in triggering programmed cell death in developing maize endosperm and is responsible for the aberrant phenotype of sh2 kernels

Although maize endosperm undergoes programmed cell death during its development, it is not known whether this developmental feature is common to cereals or whether it arose inadvertently from the selection process that resulted in the enlarged endosperm of modern maize. Examination of wheat endosperm during its development revealed that this tissue undergoes a programmed cell death that shares features with the maize program but differs in some aspects of its execution. Cell death initiated and progressed stochastically in wheat endosperm in contrast to maize where cell death initiates within the upper central endosperm and expands outward. After a peak of ethylene production during early development, wheat endosperm DNA underwent internucleosomal fragmentation that was detectable from mid to late development. The developmental onset and progression of DNA degradation was regulated by the level of ethylene production and perception. These observations suggest that programmed cell death of the endosperm and regulation of this program by ethylene is not unique to maize but that differences in the execution of the program appear to exist among cereals.

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels