Paolo A. Sabelli

The development of endosperm in grasses.

The grass seed or caryopsis originates from a monocarpellary ovary with a single ovule and contains the main storage tissue, the endosperm. For most grass crop species (i.e. cereals), the value of the crop is largely determined by the endosperm, both in quantitative and qualitative terms. The

Characterization and organization of gene families at the Gli-1 loci of bread and durum wheats by restriction fragment analysis

SummaryProbes related to γ-gliadins and to the LMW subunits of glutenin were used to determine the complexity of the Gli-1 loci, by RFLP analysis of euploid and aneuploid lines of bread wheat cv Chinese Spring and durum wheat cv Langdon. The two probes hybridised to separate sets of fragments derived from chromosomes 1 A, 1 B and 1D. The fragments related to the LMW subunit probe had a total copy number in HindIII digests of about 35 in Chinese Spring and 17 in Langdon, with more fragments derived from chromosomes 1D. The fragments hybridising to the γ-gliadin probe could be divided into two classes, based on whether they hybridised to the whole probe at high stringency or to the 3′ nonrepetitive region at moderate stringency. The fragments that failed to hybridise under these conditions were considered to be related to ω-gliadins. The fragments related to γ — and co-gliadins had total copy numbers of about 39 and 16, respectively, in HindIII digests of Chinese Spring, and about 24 and 12, respectively, in Langdon.

A Dominant Negative Mutant of Cyclin-Dependent Kinase A Reduces Endoreduplication but Not Cell Size or Gene Expression in Maize Endosperm

Cells in maize (Zea mays) endosperm undergo multiple cycles of endoreduplication, with some attaining DNA contents as high as 96C and 192C. Genome amplification begins around 10 d after pollination, coincident with cell enlargement and the onset of starch and storage protein accumulation. Although the role of endoreduplication is unclear, it is thought to provide a mechanism that increases cell size and enhances gene expression. To investigate this process, we reduced endoreduplication in transgenic maize endosperm by ectopically expressing a gene encoding a dominant negative mutant form of cyclin-dependent kinase A. This gene was regulated by the 27-kD γ-zein promoter, which restricted synthesis of the defective enzyme to the endoreduplication rather than the mitotic phase of endosperm development. Overexpression of a wild-type cyclin-dependent kinase A increased enzyme activity but had no effect on endoreduplication. By contrast, ectopic expression of the defective enzyme lowered kinase activity and reduced by half the mean C-value and total DNA content of endosperm nuclei. The lower level of endoreduplication did not affect cell size and only slightly reduced starch and storage protein accumulation. There was little difference in the level of endosperm gene expression with high and low levels of endoreduplication, suggesting that this process may not enhance transcription of genes associated with starch and storage protein synthesis.

Cyclin-Dependent Kinase Inhibitors in Maize Endosperm and Their Potential Role in Endoreduplication

Two maize (Zea mays) cyclin-dependent kinase (CDK) inhibitors, Zeama;KRP;1 and Zeama;KRP;2, were characterized and shown to be expressed in developing endosperm. Similar to the CDK inhibitors in Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), the maize proteins contain a carboxy-terminal region related to the inhibitory domain of the mammalian Cip/Kip inhibitors. Zeama;KRP;1 is present in the endosperm between 7 and 21 d after pollination, a period that encompasses the onset of endoreduplication, while the Zeama;KRP;2 protein declines during this time. Nevertheless, Zeama;KRP;1 accounts for only part of the CDK inhibitory activity that peaks coincident with the endoreduplication phase of endosperm development. In vitro assays showed that Zeama;KRP;1 and Zeama;KRP;2 are able to inhibit endosperm Cdc2-related CKD activity that associates with p13Suc1. They were also shown to specifically inhibit cyclin A1;3- and cyclin D5;1-associated CDK activities, but not cyclin B1;3/CDK. Overexpression of Zeama;KRP;1 in maize embryonic calli that ectopically expressed the wheat dwarf virus RepA protein, which counteracts retinoblastoma-related protein function, led to an additional round of DNA replication without nuclear division.

The resistance of cowpea seeds to bruchid beetles is not related to levels of cysteine proteinase inhibitors

A cDNA encoding a cysteine proteinase inhibitor was isolated from a cDNA library prepared from developing seeds of an insect-resistant line of cowpea. The sequence of the encoded protein was homologous with those of other plant cysteine endoproteinase inhibitors, and with Type 2 cystatins from animals. Southern blot analyses indicated that small gene families were present in both resistant and susceptible lines of cowpea, while northern blot analyses showed similar levels of expression. It is concluded that the levels of expression of the inhibitor do not account for the differences in insect resistance of the two lines.

The contribution of cell cycle regulation to endosperm development

Development of the seed endosperm involves several different types of coordinated cell cycle programs: acytokinetic mitosis, which produces a syncytium soon after fertilization; cellularization through the formation of modified phragmoplasts; cell proliferation, in which mitosis is coupled to cell division; and, in certain species like cereal crops, endoreduplication. Understanding the regulation of these programs and their transitions is challenging, but it has the potential to define important links between the cell cycle, cell differentiation and development, as well as provide tools for the manipulation of seed yield. A relatively large number of mutants display endosperm proliferation defects, and connections with known cell cycle genes are beginning to emerge. For example, it is becoming increasingly evident that the master cell cycle regulators, the cyclin-dependent kinases and retinoblastoma-related families, play key roles in the events leading to endosperm formation and development. Recent studies highlight cross-talk between pathways controlling the cell cycle and genomic imprinting.

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

Significance Cereal endosperm is a key source of dietary calories and raw materials for countless manufactured goods. Understanding how the cell cycle is regulated during endosperm development could lead to increased crop yield. We show that a maize Retinoblastoma-related gene, RBR1, plays a central role in regulating gene expression, endoreduplication, and the number, size, and death of endosperm cells. RBR1 is genetically coupled to Cyclin Dependent Kinase A;1 in controlling endoreduplication but not gene expression. Seeds down-regulated for RBR1 develop normally, which suggests higher-order control mechanisms regulating endosperm development that are superimposed on cell cycle regulation. 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.

Grasses like mammals? Redundancy and compensatory regulation within the retinoblastoma protein family.

The retinoblastoma (RB) protein family plays a conserved and inhibitory role incell cycle progression in higher eukaryotes. In mammals, this family includes, in additionto RB, the related (RBR) proteins p107 and p130, which appear to have both specific andredundant functions compared to those of the prototypical RB protein. Whereas mostplant species seem to possess only one RBR gene, a recent study has shown that in maizethere are two types of distinctly regulated RBR proteins, RBR1 and RBR3. Expression ofRBR3 RNA is controlled by the RBR1-E2F pathway, and it is up-regulated uponinhibition of RBR1 activity by the wheat dwarf virus RepA protein in tissue culture,indicating the presence of a specific compensatory mechanism sustaining high pocketprotein activity. Database mining and phylogenetic analyses suggest the presence of twodistinct RBR genes to be a unique feature of grasses among plants, which might help toexplain their recalcitrance to genetic transformation.

cDNA cloning and characterisation of a maize homologue of the MCM proteins required for the initiation of DNA replication.

A central question in cell cycle regulation is how DNA replication is initiated and executed only once in each cell cycle. The cell cycle-regulated assembly of specific initiation protein complexes at chromosomal origins appears to specify the initial sites and timing of DNA replication, and to restrict this process to only one round in the somatic cell cycle. Among the enzymes involved in origin activation, the MCM proteins play a conserved key role. In particular, MCM3 homologues have been shown to be components of the DNA replication licensing activity in yeast and vertebrates. In spite of our detailed knowledge of the regulation of the initiation of DNA synthesis in yeast, there is virtually no information available on the molecules involved in origin activation in higher plants. We have isolated a cDNA from maize root apices, termedROA (Replication Origin Activator), encoding a protein which shares a high degree of homology with the MCM3 subfamily of MCM proteins. Analysis of gene organisation by Southern blotting shows 2–4 copies per haploid genome of closely relatedROA sequences and the presence of further less related sequences in a multigene family. The steady-state levels ofROA mRNA are under developmental control, being relatively high in proliferative tissues such as the root apex, the developing cob and the coleoptile, and are strongly correlated with that of the histone H4 transcript.In situ hybridisation analysis in the root apex reveals thatROA mRNA expression is limited to specific subpopulations of cycling cells, which is typical of cell cycle-regulated expression. The isolation of nearly identical sequences from barley andArabidopsis by the polymerase chain reaction indicates that MCM-related proteins are conserved in higher plants.

Chromosomal location of seed storage protein genes in the genome ofDasypyrum villosum (L.) Candargy

SummaryGenes coding for glutenin-like subunits and for several prolamin subunits with electrophoretic mobilities (lactate-PAGE) corresponding to those of omega- and gamma-gliadins of wheat were located inDasypyrum villosum chromosome1V. Genes controlling four gliadinlike subunits with electrophoretic mobilities corresponding to those of alpha- and gamma-gliadins were located on the short arm of chromosome6V and on the long arm of chromosome4V. N-terminal amino acid sequences of these four components were also determined and homology with alpha-type gliadins was demonstrated. The presence of genes coding for glutenin- and gliadin-like subunits on chromosomes1V and6V demonstrates homoeology between theD. villosum chromosomes1V and6V and the chromosomes of homoeologous groups 1 and 6 in wheat. It is likely that the additional locusGli-V3 on chromosome4V originated by translocation from theGli-V2 locus.