The relative organization of genes and repetitive DNAs in complex eukaryotic genomes is not well understood. Diagnostic sequencing indicated that a 280-kilobase region containing the maize Adh1-F and u22 genes is composed primarily of retrotransposons inserted within each other. Ten retroelement families were discovered, with reiteration frequencies ranging from 10 to 30,000 copies per haploid genome. These retrotransposons accounted for more than 60 percent of the Adh1-F region and at least 50 percent of the nuclear DNA of maize. These elements were largely intact and are dispersed throughout the gene-containing regions of the maize genome.

Pre-exposure to stress may alter plants' subsequent responses by producing faster and/or stronger reactions implying that plants exercise a form of 'stress memory'. The mechanisms of plants' stress memory responses are poorly understood leaving this fundamental biological question unanswered. Here we show that during recurring dehydration stresses Arabidopsis plants display transcriptional stress memory demonstrated by an increase in the rate of transcription and elevated transcript levels of a subset of the stress-response genes (trainable genes). During recovery (watered) states, trainable genes produce transcripts at basal (preinduced) levels, but remain associated with atypically high H3K4me3 and ser5P polymerase II levels, indicating that RnA polymerase II is stalled. This is the first example of a stalled RnA polymerase II and its involvement in transcriptional memory in plants. These newly discovered phenomena might be a general feature of plant stress-response systems and could lead to novel approaches for increasing the flexibility of a plant's ability to respond to the environment.

Chromatin immunoprecipitation (ChIP) is a powerful tool for the characterization of covalent histone modifications and DNA-histone interactions in vivo. The procedure includes DNA-histone cross-linking in chromatin, shearing DNA into smaller fragments, immunoprecipitation with antibodies against the histone modifications of interest, followed by PCR identification of associated DNA sequences. In this protocol, we describe a simplified and optimized version of ChIP assay by reducing the number of experimental steps and isolation solutions and shortening preparation times. We include a nuclear isolation step before chromatin shearing, which provides a good yield of high-quality DNA resulting in at least 15 mug of DNA from each immunoprecipitated sample (from 0.2 to 0.4 g of starting tissue material) sufficient to test > or =25 genes of interest. This simpler and cost-efficient protocol has been applied for histone-modification studies of various Arabidopsis thaliana tissues and is easy to adapt for other systems as well.

Orthologous adh regions of the sorghum and maize genomes were sequenced and analyzed. Nine known or candidate genes, including adh1, were found in a 225-kilobase (kb) maize sequence. In a 78-kb space of sorghum, the nine homologues of the maize genes were identified in a colinear order, plus five additional genes. The major fraction of DNA in maize, occupying 166 kb (74%), is represented by 22 long terminal repeat (LTR) retrotransposons. About 6% of the sequence belongs to 33 miniature inverted-repeat transposable elements (MITEs), remnants of DNA transposons, 4 simple sequence repeats, and low-copy-number DNAs of unknown origin. In contrast, no LTR retroelements were detected in the orthologous sorghum region. The unconserved sorghum DNA is composed of 20 putative MITEs, transposon-like elements, 5 simple sequence repeats, and low-copy-number DNAs of unknown origin. No MITEs were discovered in the 166 kb of DNA occupied by the maize LTR retrotransposons. In both species, MITEs were found in the space between genes and inside introns, indicating specific insertion and͞or retention for these elements. Two adjacent sorghum genes, including one gene missing in maize, had colinear homologues on Arabidopsis chromosome IV, suggesting two rearrangements in the sorghum and three in the maize genome in comparison to a four-gene region of Arabidopsis. Hence, multiple small rearrangements may be present even in largely colinear genomic regions. These studies revealed a much higher degree of diversity at a microstructural level than predicted by genetic mapping studies for closely related grass species, as well as for comparisons of monocots and dicots.The grasses belong to a family of monocotyledonous angiosperms that are well differentiated morphologically from the other angiosperm families and have a single (monophyletic) origin. Their genome sizes, however, may vary a great deal between species. Thus, rice has an estimated genome size of 430 megabases, which is Ϸ11ϫ smaller than barley, 6ϫ smaller than maize, and 2ϫ smaller than sorghum. These large differences in genome sizes, coupled with differences in the degree and the nature of their investigations, have obscured some common features of grass genomic design. Recent studies comparing high-density linkage maps with DNA markers revealed extensive synteny of chromosomal segments between related species (1-5). Valuable as it is, full genome recombinational mapping of DNA markers is not an efficient approach for detecting small rearrangements. Because the available high-resolution maps based on completed nucleotide sequence are largely restricted to individual genes and their proximal neighborhoods, we are left with two obvious questions that cannot be answered at a full-genome level of analysis. These questions are, will the colinearity observed at the 2-to 20-centimorgan level, the sensitivity level of standard recombinational mapping, be preserved or will it break down at a local level (5), and what will the pattern of gene distribution be, relative to the no...

BackgroundHow plants respond to dehydration stress has been extensively researched. However, how plants respond to multiple consecutive stresses is virtually unknown. Pre-exposure to various abiotic stresses (including dehydration) may alter plants’ subsequent responses by improving resistance to future exposures. These observations have led to the concept of ‘stress memory’ implying that during subsequent exposures plants provide responses that are different from those during their first encounter with the stress. Genes that provide altered responses in a subsequent stress define the ‘memory genes’ category; genes responding similarly to each stress form the ‘non-memory’ category.ResultsUsing a genome-wide RNA-Seq approach we determine the transcriptional responses of Arabidopsis plants that have experienced multiple exposures to dehydration stress and compare them with the transcriptional behavior of plants encountering the stress for the first time. The major contribution of this study is the revealed existence of four distinct, previously unknown, transcription memory response patterns of dehydration stress genes in A.thaliana. The biological relevance for each of the four memory types is considered in the context of four overlapping strategies employed by a plant to improve its stress tolerance and/or survival: 1) increased synthesis of protective, damage-repairing, and detoxifying functions; 2) coordinating photosynthesis and growth under repetitive stress; 3) re-adjusting osmotic and ionic equilibrium to maintain homeostasis; and 4) re-adjusting interactions between dehydration and other stress/hormone regulated pathways.ConclusionsThe results reveal the unknown, hitherto, existence of four distinct transcription memory response types in a plant and provide genome-wide characterization of memory and non-memory dehydration stress response genes in A.thaliana. The transcriptional responses during repeated exposures to stress are different from known responses occurring during a single exposure. GO analyses of encoded proteins suggested implications for the cellular/organismal protective, adaptive, and survival functions encoded by the memory genes. The results add a new dimension to our understanding of plants’ responses to dehydration stress and to current models for interactions between different signaling systems when adjusting to repeated spells of water deficits.