No abstract

A cDNA, cRKIN1, encoding a putative homologue of the yeast (Saccharomyces cerevisuiae) SNFl (6), human cells (7), and Xenopus (8) are also present in pea (9) and Arabidopsis (10). All protein kinases that have been characterized in detail contain a number of key residues and conserved regions in the catalytic domain (11) but can be divided into two classes: those that phosphorylate serine/threonine residues and those that phosphorylate tyrosine residues. In the present report, we present the nucleotide sequence of a cDNA, cRKIN1,** isolated from a rye endosperm cDNA library. The cRKIN1-encoded protein contains all the invariant residues and conserved domains characteristic ofeukaryotic protein-serine/threonine kinases. It is particularly similar to the product ofthe SNFJ gene of yeast (Saccharomyces cerevisiae) (12), a protein affecting global regulation of carbon metabolism, and the expression of cRKIN1 in yeast snfl mutants restores SNFJ function.

Reactive oxygen species are common causes of cellular damages in all aerobic organisms. In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H202 stress. To identify genes involved in counteracting oxidative stress in plants, we transformed a AoxyR mutant of E. coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the AoxyR mutant to grow in the presence of H202. Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family. The complemented mutant showed higher catalase activity. mRNA expression of the annexin gene in A. thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H202 stress or salicylic acid. Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H202 stress.

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

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.

Retinoblastoma-related (RBR) proteins regulate cell division in higher eukaryotes by controlling the adenovirus E2 promoter binding factor (E2F)͞dimerization partner (DP) family of transcription factors that regulate expression of many genes involved in cell-cycle progression. We identified a previously undescribed member of the maize RBR family, RBR3, which has the characteristic structure and binding activities of pocket proteins, where interaction depends on a LxCxE motif in the partner proteins and a critical cysteine within the B pocket domain. Like other RBR proteins, RBR3 appears to be regulated by phosphorylation mediated by cyclindependent kinases. During endosperm development, RBR3 expression is restricted to the mitotic stage preceding the onset of endoreduplication. This finding suggests a role distinct from RBR1, which is constitutively expressed. Two sites in the RBR3 promoter bind to complexes containing maize E2F1 and DP proteins. Expression of wheat dwarf virus RepA protein, which blocks RBR1 activity and stimulates cell proliferation, dramatically up-regulates RBR3, but not RBR1, RNA in embryogenic maize calli. The results indicate that RBR3 expression is controlled by RBR1 through the activity of E2F͞DP and that RBR3 is the maize equivalent of mammalian p107. Furthermore, maize and related grasses might have evolved a compensatory mechanism among distinct types of RBR proteins to ensure robust control of pocket protein activity.cell cycle ͉ pocket protein ͉ RepA S pecific gene expression programs are key targets for cell-cycle control. As cells advance through G 1 and S phase, they sequentially up-regulate batteries of genes involved in licensing of replication origins, synthesis of G 1 ͞S-phase-specific cyclins (Cycs) and Cyc-dependent kinases (CDKs), and DNA synthesis. The adenovirus E2 promoter binding factor (E2F) family of transcription factors controls the expression of many genes required for entry into and execution of S-phase and cell-cycle progression (reviewed in refs. 1-4). Most E2F proteins require dimerization with a distantly related dimerization partner (DP) protein for activity. In humans, at least eight E2F family members have been identified that can be functionally grouped into transcriptional activators or repressors (reviewed in refs. 4 and 5), and a similar family of E2F proteins is present in plants (6-8).The retinoblastoma-related (RBR) family of proteins (RB, p107, and p130 in mammals) primarily represses the G 1 ͞S-phase transition of the cell cycle by inhibiting, directly (through masking of the E2F transactivation domain) or indirectly (by recruiting different chromatin-remodeling complexes and thereby silencing specific chromatin regions), the E2F͞DP family of transcription factors (reviewed in refs. 9 and 10). Collectively, the activity of RBR proteins results in inhibition of the expression of E2F-regulated genes in G 1 , effectively preventing the transition into S phase. The consensus understanding from many studies in mammals indicates that from late G 1 , in...

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 MAINTE-NANCE 2-7 gene family and PROLIFERATING CELL NUCLEAR ANTI-GEN, 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 downregulation, 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/organlevel regulation of endosperm/seed homeostasis. seed development | endocycle T he seed endosperm is a triploid tissue resulting from the fusion of one haploid sperm nucleus with the diploid central cell nucleus within the female gametophyte. Development of the endosperm in flowering plants is characterized by acytokinetic mitoses of the primary endosperm nucleus, resulting in a syncytium, cellularization of syncytial nuclear domains, and cell proliferation through mitotic activity that is coupled to cell division (1, 2). Additionally, in the Poaceae (grass) family, the endosperm undergoes a rapid growth phase that coincides with accumulation of storage compounds, such as starch and storage proteins, during a specialized type of cell cycle known as endoreduplication. Endoreduplication is characterized by one or more rounds of DNA synthesis in the absence of mitosis, resulting in polyploid cells (3-5). Endoreduplication is highly correlated with cell size in many plant and animal tissues, but its role in endosperm development has not been established. Upon completion of endoreduplication and storage metabolite synthesis, cereal endosperm cells undergo programmed cell death (PCD), resulting in extensive DNA degradation (5, 6). In maize (Zea mays L.), endosperm cells transition from a mitotic to an endore...

Probes 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.