Abed Chaudhury

pEmu: an improved promoter for gene expression in cereal cells

SummaryA recombinant promoter, pEmu, has been constructed to give a high level of gene expression in monocots. It is based on a truncated maize Adh1 promoter, with multiple copies of the Anaerobic Responsive Element from the maize Adh1 gene and ocs-elements from the octopine synthase gene of Agrobacterium tumefaciens. The pEmu promoter was one of 12 different promoter constructs that were linked to the β-glucuronidase (GUS) marker gene. Promoter activity was measured 48 h after introduction of the constructs into protoplasts of five different monocot species [wheat, maize, rice, einkorn (Triticum monococcum), and Lolium multiflorum] and one dicot (Nicotiana plumbaginifolia). In suspension cell protoplasts, the most highly expressing construct (pEmuGN) gave 10- to 50-fold higher expression than the CaMV 35S promoter in all the monocot species. The pEmu promoter should be valuable where a high level of gene expression is required in monocots. The pEmu promoter showed instability in several widely used Escherichia coli strains but was stable in a recA, recD strain AC001, which is described. Another construct, p4OCSΔ35SIGN, gave a tenfold increase in expression over the CaMV 35S promoter in dicot (Nicotiana plumbaginifolia) protoplasts.

The Arabidopsis AMP1 Gene Encodes a Putative Glutamate Carboxypeptidase

Arabidopsis amp1 mutants show pleiotropic phenotypes, including altered shoot apical meristems, increased cell proliferation, polycotyly, constitutive photomorphogenesis, early flowering time, increased levels of endogenous cytokinin, and increased cyclin cycD3 expression. We have isolated the AMP1 gene by map-based cloning. The AMP1 cDNA encodes a 706;–amino acid polypeptide with significant similarity to glutamate carboxypeptidases. The AMP1 mRNA was expressed in all tissues examined, with higher expression in roots, stems, inflorescences, and siliques. Microarray analysis identified four mRNA species with altered expression in two alleles of amp1, including upregulation of CYP78A5, which has been shown to mark the shoot apical meristem boundary. The similarity of the AMP1 protein to glutamate carboxypeptidases, and in particular to N-acetyl α-linked acidic dipeptidases, suggests that the AMP1 gene product modulates the level of a small signaling molecule that acts to regulate a number of aspects of plant development, in particular the size of the apical meristem.

Expression, Imprinting, and Evolution of Rice Homologs of the Polycomb Group Genes

Polycomb group proteins (PcG) play important roles in epigenetic regulation of gene expression. Some core PcG proteins, such as Enhancer of Zeste (E(z)), Suppressor of Zeste (12) (Su(z)12), and Extra Sex Combs (ESC), are conserved in plants. The rice genome contains two E(z)-like genes, OsiEZ1 and OsCLF, two homologs of Su(z)12, OsEMF2a and OsEMF2b, and two ESC-like genes, OsFIE1 and OsFIE2. OsFIE1 is expressed only in endosperm; the maternal copy is expressed while the paternal copy is not active. Other rice PcG genes are expressed in a wide range of tissues and are not imprinted in the endosperm. The two E(z)-like genes appear to have duplicated before the separation of the dicots and monocots; the two homologs of Su(z)12 possibly duplicated during the evolution of the Gramineae and the two ESC-like genes are likely to have duplicated in the ancestor of the grasses. No homologs of the Arabidopsis seed-expressed PcG genes MEA and FIS2 were identified in the rice genome. We have isolated T-DNA insertion lines in the rice homologs of three PcG genes. There is no autonomous endosperm development in these T-DNA insertion lines. One line with a T-DNA insertion in OsEMF2b displays pleiotropic phenotypes including altered flowering time and abnormal flower organs, suggesting important roles in rice development for this gene.

The VQ motif protein IKU1 regulates endosperm growth and seed size in Arabidopsis

Arabidopsis seed size is regulated by the IKU pathway that includes IKU2 (a leucine-rich repeat kinase) and MINI3 (a WRKY transcription factor). We report the cloning of the IKU1 (At2g35230) gene. iku1 mutants cause reduced endosperm growth and the production of small seeds. IKU1 encodes a protein containing a VQ motif, which is a motif specific to plants. IKU1 is expressed in the early endosperm and its progenitor, the central cell. Restoration of IKU1 function in the endosperm is sufficient to rescue seed size. A genomic construct carrying mutations in the VQ motif failed to complement the iku1 mutation, suggesting an essential role for the VQ motif. IKU1 interacts with MINI3 in the yeast two-hybrid system, consistent with an IKU1 function in the IKU-MINI pathway. Our data support the proposition that endosperm development is an important determinant of seed size.

Parental memories shape seeds.

It is ten years since imprinting was first demonstrated in Arabidopsis, following the realization, five years earlier, that some genetic controls of seed development did not conform to Mendelian inheritance. Sixteen imprinted genes have since been identified in maize and Arabidopsis and these are expressed primarily in the endosperm, which nurtures embryo development. Imprinting results from the regulation of transcriptional silencing by DNA methylation or by Polycomb Group complex-mediated histone methylation. Here we review recent studies suggesting that imprinting results from global epigenetic changes that occur during female gametogenesis. We also discuss why imprinting has evolved and what its biological functions might be.

Genetic control of male fertility in Arabidopsis thaliana : structural analysis of premeiotic developmental mutants

We have taken a mutational approach to identify genes important for male fertility in Arabidopsis thaliana and have isolated a number of nuclear male/ sterile mutants in which vegetative growth and female fertility are not altered. Here we describe detailed developmental analyses of four mutants, each of which defines a complementation group and has a distinct developmental end point. All four mutants represent premeiotic developmental lesions. In ms3, tapetum and middle layer hypertrophy result in the degeneration of microsporocytes. In ms4, microspore dyads persist for most of anther development as a result of impaired meiotic division. In ms5, degeneration occurs in all anther cells at an early stage of development. In ms15, both the tapetum and microsporocytes degenerate early in anther development. Each of these mutants had shorter filaments and a greater number of inflorescences than congenic male-fertile plants. The differences in the developmental phenotypes of these mutants, together with the non-allelic nature of the mutations indicate that four different genes important for pollen development, have been identified.

DNA Methylation Causes Predominant Maternal Controls of Plant Embryo Growth

The parental conflict hypothesis predicts that the mother inhibits embryo growth counteracting growth enhancement by the father. In plants the DNA methyltransferase MET1 is a central regulator of parentally imprinted genes that affect seed growth. However the relation between the role of MET1 in imprinting and its control of seed size has remained unclear. Here we combine cytological, genetic and statistical analyses to study the effect of MET1 on seed growth. We show that the loss of MET1 during male gametogenesis causes a reduction of seed size, presumably linked to silencing of the paternal allele of growth enhancers in the endosperm, which nurtures the embryo. However, we find no evidence for a similar role of MET1 during female gametogenesis. Rather, the reduction of MET1 dosage in the maternal somatic tissues causes seed size increase. MET1 inhibits seed growth by restricting cell division and elongation in the maternal integuments that surround the seed. Our data demonstrate new controls of seed growth linked to the mode of reproduction typical of flowering plants. We conclude that the regulation of embryo growth by MET1 results from a combination of predominant maternal controls, and that DNA methylation maintained by MET1 does not orchestrate a parental conflict.

UBIQUITIN-SPECIFIC PROTEASE 26 Is Required for Seed Development and the Repression of PHERES1 in Arabidopsis

The Arabidopsis mutant Atubp26 initiates autonomous endosperm at a frequency of ∼1% in the absence of fertilization and develops arrested seeds at a frequency of ∼65% when self-pollinated. These phenotypes are similar to those of the FERTILIZATION INDEPENDENT SEED (FIS) class mutants, mea, fis2, fie, and Atmsi1, which also show development of the central cell into endosperm in the absence of fertilization and arrest of the embryo following fertilization. Atubp26 results from a T-DNA insertion in the UBIQUITIN-SPECIFIC PROTEASE gene AtUBP26, which catalyzes deubiquitination of histone H2B and is required for heterochromatin silencing. The paternal copy of AtUBP26 is able to complement the loss of function of the maternal copy in postfertilization seed development. This contrasts to the fis class mutants where the paternal FIS copy does not rescue aborted seeds. As in the fis class mutants, the Polycomb group (PcG) complex target gene PHERES1 (PHE1) is expressed at higher levels in Atubp26 ovules than in wild type; there is a lower level of H3K27me3 at the PHE1 locus. The phenotypes suggest that AtUBP26 is required for normal seed development and the repression of PHE1.

Genetic and epigenetic processes in seed development

Seed development has emerged as an important area of research in plant development. Recent research has highlighted the divergent reproductive strategies of the male and female genomes and interaction between genetic and epigenetic control mechanisms. Isolation of genes involved in embryo and endosperm development is leading to an understanding of the regulation of these processes at the molecular level. A thorough grasp of these processes will not only illuminate an important area of plant development but will also have an impact on agronomy by helping to facilitate food production. An understanding of seed development is also likely to clarify the molecular mechanisms of apomixis, a fascinating process of asexual seed production present in many plants.

Transgene Expression and Transgene-induced Silencing in Diploid and Autotetraploid Arabidopsis

Previous studies have suggested that transgene expression in plants can be affected by ploidy. Here we show that three different transgenes, a reporter transgene, an antisense transgene, and a hairpin RNA (hpRNA) transgene, are all expressed at a lower level in autotetraploid (4n) than in diploid (2n) Arabidopsis. RNA silencing of two endogenous genes was induced by the antisense and hpRNA transgenes and this silencing is significantly less effective in 4n than in 2n Arabidopsis; furthermore, the reduced silencing in 4n Arabidopsis correlated with reduced accumulation of silencing-inducer RNAs. Methylation analysis both of independent 2n and 4n transgenic lines and of 2n and 4n progeny derived from the same 3n transgenic parent, indicated that transgenes are more methylated in 4n than 2n Arabidopsis. These results suggest that transgenes are transcriptionally repressed in the 4n background, resulting in expression levels lower than in the 2n background. Transgenes designed to silence endogenous genes express lower concentrations of silencing-inducer RNAs in 4n Arabidopsis plants, resulting in less effective silencing of target genes than in 2n Arabidopsis plants.