Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the Natural Resistance Associated-Macrophage Protein3natural Resistance Associated-Macrophage Protein4 Double Mutant

To improve seed iron (Fe) content and bioavailability, it is crucial to decipher the mechanisms that control Fe storage during seed development. In Arabidopsis (Arabidopsis thaliana) seeds, most Fe is concentrated in insoluble precipitates, with phytate in the vacuoles of cells surrounding the vasculature of the embryo. NATURAL RESISTANCE ASSOCIATED-MACROPHAGE PROTEIN3 (AtNRAMP3) and AtNRAMP4 function redundantly in Fe retrieval from vacuoles during germination. When germinated under Fe-deficient conditions, development of the nramp3nramp4 double mutant is arrested as a consequence of impaired Fe mobilization. To identify novel genes involved in seed Fe homeostasis, we screened an ethyl methanesulfonate-mutagenized population of nramp3nramp4 seedlings for mutations suppressing their phenotypes on low Fe. Here, we report that, among the suppressors, two independent mutations in the VACUOLAR IRON TRANSPORTER1 (AtVIT1) gene caused the suppressor phenotype. The AtVIT1 transporter is involved in Fe influx into vacuoles of endodermal and bundle sheath cells. This result establishes a functional link between Fe loading in vacuoles by AtVIT1 and its remobilization by AtNRAMP3 and AtNRAMP4. Moreover, analysis of subcellular Fe localization indicates that simultaneous disruption of AtVIT1, AtNRAMP3, and AtNRAMP4 limits Fe accumulation in vacuolar globoids

Identification of Cis-Acting Promoter Elements in Cold- and Dehydration-Induced Transcriptional Pathways in Arabidopsis, Rice, and Soybean

The genomes of three plants, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and soybean (Glycine max), have been sequenced, and their many genes and promoters have been predicted. In Arabidopsis, cis-acting promoter elements involved in cold- and dehydration-responsive gene expression have been extensively analysed; however, the characteristics of such cis-acting promoter sequences in cold- and dehydration-inducible genes of rice and soybean remain to be clarified. In this study, we performed microarray analyses using the three species, and compared characteristics of identified cold- and dehydration-inducible genes. Transcription profiles of the cold- and dehydration-responsive genes were similar among these three species, showing representative upregulated (dehydrin/LEA) and downregulated (photosynthesis-related) genes. All (46 = 4096) hexamer sequences in the promoters of the three species were investigated, revealing the frequency of conserved sequences in cold- and dehydration-inducible promoters. A core sequence of the abscisic acid-responsive element (ABRE) was the most conserved in dehydration-inducible promoters of all three species, suggesting that transcriptional regulation for dehydration-inducible genes is similar among these three species, with the ABRE-dependent transcriptional pathway. In contrast, for cold-inducible promoters, the conserved hexamer sequences were diversified among these three species, suggesting the existence of diverse transcriptional regulatory pathways for cold-inducible genes among the species

Circadian control of abscisic acid biosynthesis and signalling pathways revealed by genome-wide analysis of LHY binding targets

The LATE ELONGATED HYPOCOTYL (LHY) transcription factor functions as part of the oscillatory mechanism of the Arabidopsis circadian clock. This paper reports the genome‐wide analysis of its binding targets and reveals a role in the control of abscisic acid (ABA) biosynthesis and downstream responses. LHY directly repressed expression of 9‐cis‐epoxycarotenoid dioxygenase enzymes, which catalyse the rate‐limiting step of ABA biosynthesis. This suggested a mechanism for the circadian control of ABA accumulation in wild‐type plants. Consistent with this hypothesis, ABA accumulated rhythmically in wild‐type plants, peaking in the evening. LHY‐overexpressing plants had reduced levels of ABA under drought stress, whereas loss‐of‐function mutants exhibited an altered rhythm of ABA accumulation. LHY also bound the promoter of multiple components of ABA signalling pathways, suggesting that it may also act to regulate responses downstream of the hormone. LHY promoted expression of ABA‐responsive genes responsible for increased tolerance to drought and osmotic stress but alleviated the inhibitory effect of ABA on seed germination and plant growth. This study reveals a complex interaction between the circadian clock and ABA pathways, which is likely to make an important contribution to plant performance under drought and osmotic stress conditions

Synergistic repression of the embryonic programme by SET DOMAIN GROUP 8 and EMBRYONIC FLOWER 2 in Arabidopsis seedlings

The seed maturation programme occurs only during the late phase of embryo development, and repression of the maturation genes is pivotal for seedling development. However, mechanisms that repress the expression of this programme in vegetative tissues are not well understood. A genetic screen was performed for mutants that express maturation genes in leaves. Here, it is shown that mutations affecting SDG8 (SET DOMAIN GROUP 8), a putative histone methyltransferase, cause ectopic expression of a subset of maturation genes in leaves. Further, to investigate the relationship between SDG8 and the Polycomb Group (PcG) proteins, which are known to repress many developmentally important genes including seed maturation genes, double mutants were made and formation of somatic embryos was observed on mutant seedlings with mutations in both SDG8 and EMF2 (EMBRYONIC FLOWER 2). Analysis of histone methylation status at the chromatin sites of a number of maturation loci revealed a synergistic effect of emf2 and sdg8 on the deposition of the active histone mark which is the trimethylation of Lys4 on histone 3 (H3K4me3). This is consistent with high expression of these genes and formation of somatic embryos in the emf2 sdg8 double mutants. Interestingly, a double mutant of sdg8 and vrn2 (vernalization2), a paralogue of EMF2, grew and developed normally to maturity. These observations demonstrate a functional cooperative interplay between SDG8 and an EMF2-containing PcG complex in maintaining vegetative cell identity by repressing seed genes to promote seedling development. The work also indicates the functional specificities of PcG complexes in Arabidopsis

Overexpression of a Common Wheat Gene TaSnRK2.8 Enhances Tolerance to Drought, Salt and Low Temperature in Arabidopsis

Drought, salinity and low temperatures are major factors limiting crop productivity and quality. Sucrose non-fermenting1-related protein kinase 2 (SnRK2) plays a key role in abiotic stress signaling in plants. In this study, TaSnRK2.8, a SnRK2 member in wheat, was cloned and its functions under multi-stress conditions were characterized. Subcellular localization showed the presence of TaSnRK2.8 in the cell membrane, cytoplasm and nucleus. Expression pattern analyses in wheat revealed that TaSnRK2.8 was involved in response to PEG, NaCl and cold stresses, and possibly participates in ABA-dependent signal transduction pathways. To investigate its role under various environmental stresses, TaSnRK2.8 was transferred to Arabidopsis under control of the CaMV-35S promoter. Overexpression of TaSnRK2.8 resulted in enhanced tolerance to drought, salt and cold stresses, further confirmed by longer primary roots and various physiological characteristics, including higher relative water content, strengthened cell membrane stability, significantly lower osmotic potential, more chlorophyll content, and enhanced PSII activity. Meanwhile, TaSnRK2.8 plants had significantly lower total soluble sugar levels under normal growing conditions, suggesting that TaSnRK2.8 might be involved in carbohydrate metabolism. Moreover, the transcript levels of ABA biosynthesis (ABA1, ABA2), ABA signaling (ABI3, ABI4, ABI5), stress-responsive genes, including two ABA-dependent genes (RD20A, RD29B) and three ABA-independent genes (CBF1, CBF2, CBF3), were generally higher in TaSnRK2.8 plants than in WT/GFP controls under normal/stress conditions. Our results suggest that TaSnRK2.8 may act as a regulatory factor involved in a multiple stress response pathways

Direct targets of the transcription factors ABA-Insensitive(ABI)4 and ABI5 reveal synergistic action by ABI4 and several bZIP ABA response factors

The plant hormone abscisic acid (ABA) is a key regulator of seed development. In addition to promoting seed maturation, ABA inhibits seed germination and seedling growth. Many components involved in ABA response have been identified, including the transcription factors ABA insensitive (ABI)4 and ABI5. The genes encoding these factors are expressed predominantly in developing and mature seeds, and are positive regulators of ABA mediated inhibition of seed germination and growth. The direct effects of ABI4 and ABI5 in ABA response remain largely undefined. To address this question, plants over-expressing ABI4 or ABI5 were used to allow identification of direct transcriptional targets. Ectopically expressed ABI4 and ABI5 conferred ABA-dependent induction of slightly over 100 genes in 11 day old plants. In addition to effector genes involved in seed maturation and reserve storage, several signaling proteins and transcription factors were identified as targets of ABI4 and/or ABI5. Although only 12% of the ABA- and ABI-dependent transcriptional targets were induced by both ABI factors in 11 day old plants, 40% of those normally expressed in seeds had reduced transcript levels in both abi4 and abi5 mutants. Surprisingly, many of the ABI4 transcriptional targets do not contain the previously characterized ABI4 binding motifs, the CE1 or S box, in their promoters, but some of these interact with ABI4 in electrophoretic mobility shift assays, suggesting that sequence recognition by ABI4 may be more flexible than known canonical sequences. Yeast one-hybrid assays demonstrated synergistic action of ABI4 with ABI5 or related bZIP factors in regulating these promoters, and mutant analyses showed that ABI4 and these bZIPs share some functions in plants

Distinct expression patterns of two Arabidopsis phytocystatin genes, AtCYS1 and AtCYS2, during development and abiotic stresses

The phytocystatins of plants are members of the cystatin superfamily of proteins, which are potent inhibitors of cysteine proteases. The Arabidopsis genome encodes seven phytocystatin isoforms (AtCYSs) in two distantly related AtCYS gene clusters. We selected AtCYS1 and AtCYS2 as representatives for each cluster and then generated transgenic plants expressing the GUS reporter gene under the control of each gene promoter. These plants were used to examine AtCYS expression at various stages of plant development and in response to abiotic stresses. Histochemical analysis of AtCYS1 promoter- and AtCYS2 promoter-GUS transgenic plants revealed that these genes have similar but distinct spatial and temporal expression patterns during normal development. In particular, AtCYS1 was preferentially expressed in the vascular tissue of all organs, whereas AtCYS2 was expressed in trichomes and guard cells in young leaves, caps of roots, and in connecting regions of the immature anthers and filaments and the style and stigma in flowers. In addition, each AtCYS gene has a unique expression profile during abiotic stresses. High temperature and wounding stress enhanced the expression of both AtCYS1 and AtCYS2, but the temporal and spatial patterns of induction differed. From these data, we propose that these two AtCYS genes play important, but distinct, roles in plant development and stress responses