Auxin-sensitive Aux/IAA proteins mediate drought tolerance in Arabidopsis by regulating glucosinolate levels.

A detailed understanding of abiotic stress tolerance in plants is essential to provide food security in the face of increasingly harsh climatic conditions. Glucosinolates (GLSs) are secondary metabolites found in the Brassicaceae that protect plants from herbivory and pathogen attack. Here we report that in Arabidopsis, aliphatic GLS levels are regulated by the auxin-sensitive Aux/IAA repressors IAA5, IAA6, and IAA19. These proteins act in a transcriptional cascade that maintains expression of GLS levels when plants are exposed to drought conditions. Loss of IAA5/6/19 results in reduced GLS levels and decreased drought tolerance. Further, we show that this phenotype is associated with a defect in stomatal regulation. Application of GLS to the iaa5,6,19 mutants restores stomatal regulation and normal drought tolerance. GLS action is dependent on the receptor kinase GHR1, suggesting that GLS may signal via reactive oxygen species. These results provide a novel connection between auxin signaling, GLS levels and drought response

CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination

Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases

BACKGROUND: In Arabidopsis, ETO1 (ETHYLENE-OVERPRODUCER1) is a negative regulator of ethylene evolution by interacting with AtACS5, an isoform of the rate-limiting enzyme, 1-aminocyclopropane-1-carboxylate synthases (ACC synthase or ACS), in ethylene biosynthetic pathway. ETO1 directly inhibits the enzymatic activity of AtACS5. In addition, a specific interaction between ETO1 and AtCUL3, a constituent of a new type of E3 ubiquitin ligase complex, suggests the molecular mechanism in promoting AtACS5 degradation by the proteasome-dependent pathway. Because orthologous sequences to ETO1 are found in many plant species including tomato, we transformed tomato with Arabidopsis ETO1 to evaluate its ability to suppress ethylene production in tomato fruits. RESULTS: Transgenic tomato lines that overexpress Arabidopsis ETO1 (ETO1-OE) did not show a significant delay of fruit ripening. So, we performed yeast two-hybrid assays to investigate potential heterologous interaction between ETO1 and three isozymes of ACC synthases from tomato. In the yeast two-hybrid system, ETO1 interacts with LE-ACS3 as well as AtACS5 but not with LE-ACS2 or LE-ACS4, two major isozymes whose gene expression is induced markedly in ripening fruits. According to the classification of ACC synthases, which is based on the C-terminal amino acid sequences, both LE-ACS3 and AtACS5 are categorized as type 2 isozymes and possess a consensus C-terminal sequence. In contrast, LE-ACS2 and LE-ACS4 are type 1 and type 3 isozymes, respectively, both of which do not possess this specific C-terminal sequence. Yeast two-hybrid analysis using chimeric constructs between LE-ACS2 and LE-ACS3 revealed that the type-2-ACS-specific C-terminal tail is required for interaction with ETO1. When treated with auxin to induce LE-ACS3, seedlings of ETO1-OE produced less ethylene than the wild type, despite comparable expression of the LE-ACS3 gene in the wild type. CONCLUSION: These results suggest that ETO1 family proteins specifically interact with and negatively regulate type 2 ACC synthases. Our data also show that Arabidopsis ETO1 can regulate type 2 ACS in a heterologous plant, tomato

Effects of Aneuploidy on Genome Structure, Expression, and Interphase Organization in Arabidopsis thaliana

Aneuploidy refers to losses and/or gains of individual chromosomes from the normal chromosome set. The resulting gene dosage imbalance has a noticeable affect on the phenotype, as illustrated by aneuploid syndromes, including Down syndrome in humans, and by human solid tumor cells, which are highly aneuploid. Although the phenotypic manifestations of aneuploidy are usually apparent, information about the underlying alterations in structure, expression, and interphase organization of unbalanced chromosome sets is still sparse. Plants generally tolerate aneuploidy better than animals, and, through colchicine treatment and breeding strategies, it is possible to obtain inbred sibling plants with different numbers of chromosomes. This possibility, combined with the genetic and genomics tools available for Arabidopsis thaliana, provides a powerful means to assess systematically the molecular and cytological consequences of aberrant numbers of specific chromosomes. Here, we report on the generation of Arabidopsis plants in which chromosome 5 is present in triplicate. We compare the global transcript profiles of normal diploids and chromosome 5 trisomics, and assess genome integrity using array comparative genome hybridization. We use live cell imaging to determine the interphase 3D arrangement of transgene-encoded fluorescent tags on chromosome 5 in trisomic and triploid plants. The results indicate that trisomy 5 disrupts gene expression throughout the genome and supports the production and/or retention of truncated copies of chromosome 5. Although trisomy 5 does not grossly distort the interphase arrangement of fluorescent-tagged sites on chromosome 5, it may somewhat enhance associations between transgene alleles. Our analysis reveals the complex genomic changes that can occur in aneuploids and underscores the importance of using multiple experimental approaches to investigate how chromosome numerical changes condition abnormal phenotypes and progressive genome instability

Circadian Oscillations of Protein-Coding and Regulatory RNAs in a Highly Dynamic Mammalian Liver Epigenome

SummaryIn the mouse liver, circadian transcriptional rhythms are necessary for metabolic homeostasis. Whether dynamic epigenomic modifications are associated with transcript oscillations has not been systematically investigated. We found that several antisense RNA, lincRNA, and microRNA transcripts also showed circadian oscillations in adult mouse livers. Robust transcript oscillations often correlated with rhythmic histone modifications in promoters, gene bodies, or enhancers, although promoter DNA methylation levels were relatively stable. Such integrative analyses identified oscillating expression of an antisense transcript (asPer2) to the gene encoding the circadian oscillator component Per2. Robust transcript oscillations often accompanied rhythms in multiple histone modifications and recruitment of multiple chromatin-associated clock components. Coupling of cycling histone modifications with nearby oscillating transcripts thus established a temporal relationship between enhancers, genes, and transcripts on a genome-wide scale in a mammalian liver. The results offer a framework for understanding the dynamics of metabolism, circadian clock, and chromatin modifications involved in metabolic homeostasis

The phosphatase laforin crosses evolutionary boundaries and links carbohydrate metabolism to neuronal disease

Lafora disease (LD) is a progressive myoclonic epilepsy resulting in severe neurodegeneration followed by death. A hallmark of LD is the accumulation of insoluble polyglucosans called Lafora bodies (LBs). LD is caused by mutations in the gene encoding the phosphatase laforin, which reportedly exists solely in vertebrates. We utilized a bioinformatics screen to identify laforin orthologues in five protists. These protists evolved from a progenitor red alga and synthesize an insoluble carbohydrate whose composition closely resembles LBs. Furthermore, we show that the kingdom Plantae, which lacks laforin, possesses a protein with laforin-like properties called starch excess 4 (SEX4). Mutations in the Arabidopsis thaliana SEX4 gene results in a starch excess phenotype reminiscent of LD. We demonstrate that Homo sapiens laforin complements the sex4 phenotype and propose that laforin and SEX4 are functional equivalents. Finally, we show that laforins and SEX4 dephosphorylate a complex carbohydrate and form the only family of phosphatases with this activity. These results provide a molecular explanation for the etiology of LD

JAZ2 controls stomata dynamics during bacterial invasion

Coronatine (COR) facilitates entry of bacteria into the plant apoplast by stimulating stomata opening. COR-induced signaling events at stomata remain unclear. We found that the COR and jasmonate isoleucine (JA-Ile) co-receptor JAZ2 is constitutively expressed in guard cells and modulates stomatal dynamics during bacterial invasion. We analyzed tissue expression patterns of AtJAZ genes and measured stomata opening and pathogen resistance in loss- and gain-of-function mutants. Arabidopsis jaz2 mutants are partially impaired in pathogen-induced stomatal closing and more susceptible to Pseudomonas. Gain-of-function mutations in JAZ2 prevent stomatal reopening by COR and are highly resistant to bacterial penetration. The JAZ2 targets MYC2, MYC3 and MYC4 directly regulate the expression of ANAC19, ANAC55 and ANAC72 to modulate stomata aperture. Due to the antagonistic interactions between the salicylic acid (SA) and JA defense pathways, efforts to increase resistance to biotrophs result in enhanced susceptibility to necrotrophs, and vice versa. Remarkably, dominant jaz2Δjas mutants are resistant to Pseudomonas syringae but retain unaltered resistance against necrotrophs. Our results demonstrate the existence of a COI1-JAZ2-MYC2,3,4-ANAC19,55,72 module responsible for the regulation of stomatal aperture that is hijacked by bacterial COR to promote infection. They also provide novel strategies for crop protection against biotrophs without compromising resistance to necrotrophs

Exploring the transcriptional landscape of plant circadian rhythms using genome tiling arrays

BACKGROUND Organisms are able to anticipate changes in the daily environment with an internal oscillator know as the circadian clock. Transcription is an important mechanism in maintaining these oscillations. Here we explore, using whole genome tiling arrays, the extent of rhythmic expression patterns genome-wide, with an unbiased analysis of coding and noncoding regions of the Arabidopsis genome. RESULTS As in previous studies, we detected a circadian rhythm for approximately 25% of the protein coding genes in the genome. With an unbiased interrogation of the genome, extensive rhythmic introns were detected predominantly in phase with adjacent rhythmic exons, creating a transcript that, if translated, would be expected to produce a truncated protein. In some cases, such as the MYB transcription factor AT2G20400, an intron was found to exhibit a circadian rhythm while the remainder of the transcript was otherwise arrhythmic. In addition to several known noncoding transcripts, including microRNA, trans-acting short interfering RNA, and small nucleolar RNA, greater than one thousand intergenic regions were detected as circadian clock regulated, many of which have no predicted function, either coding or noncoding. Nearly 7% of the protein coding genes produced rhythmic antisense transcripts, often for genes whose sense strand was not similarly rhythmic. CONCLUSIONS This study revealed widespread circadian clock regulation of the Arabidopsis genome extending well beyond the protein coding transcripts measured to date. This suggests a greater level of structural and temporal dynamics than previously known

Meiotic Transmission of an In Vitro–Assembled Autonomous Maize Minichromosome

Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7–190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms