Hironaka Tsukagoshi

Transcriptional Regulation of ROS Controls Transition from Proliferation to Differentiation in the Root

The balance between cellular proliferation and differentiation is a key aspect of development in multicellular organisms. Using high-resolution expression data from the Arabidopsis root, we identified a transcription factor, UPBEAT1 (UPB1), that regulates this balance. Genomewide expression profiling coupled with ChIP-chip analysis revealed that UPB1 directly regulates the expression of a set of peroxidases that modulate the balance of reactive oxygen species (ROS) between the zones of cell proliferation and the zone of cell elongation where differentiation begins. Disruption of UPB1 activity alters this ROS balance, leading to a delay in the onset of differentiation. Modulation of either ROS balance or peroxidase activity through chemical reagents affects the onset of differentiation in a manner consistent with the postulated UPB1 function. This pathway functions independently of auxin and cytokinin plant hormonal signaling. Comparison to ROS-regulated growth control in animals suggests that a similar mechanism is used in plants and animals.

The bHLH Transcription Factor POPEYE Regulates Response to Iron Deficiency in Arabidopsis Roots

Iron deficiency induces a range of physiological responses that are controlled by transcriptional alterations concentrated in the root pericycle. The transcriptional regulator POPEYE regulates many of these responses possibly through interaction with iron deficiency response protein ILR3 and the putative E3 ligase protein BRUTUS. Global population increases and climate change underscore the need for better comprehension of how plants acquire and process nutrients such as iron. Using cell type–specific transcriptional profiling, we identified a pericycle-specific iron deficiency response and a bHLH transcription factor, POPEYE (PYE), that may play an important role in this response. Functional analysis of PYE suggests that it positively regulates growth and development under iron-deficient conditions. Chromatin immunoprecipitation-on-chip analysis and transcriptional profiling reveal that PYE helps maintain iron homeostasis by regulating the expression of known iron homeostasis genes and other genes involved in transcription, development, and stress response. PYE interacts with PYE homologs, including IAA–Leu Resistant3 (ILR3), another bHLH transcription factor that is involved in metal ion homeostasis. Moreover, ILR3 interacts with a third protein, BRUTUS (BTS), a putative E3 ligase protein, with metal ion binding and DNA binding domains, which negatively regulates the response to iron deficiency. PYE and BTS expression is also tightly coregulated. We propose that interactions among PYE, PYE homologs, and BTS are important for maintaining iron homeostasis under low iron conditions.

Two B3 domain transcriptional repressors prevent sugar-inducible expression of seed maturation genes in Arabidopsis seedlings.

During development of plant seeds, embryos import nutrients and store massive amounts of reserves. Seed reserves are rapidly degraded and mobilized to support seedling development after germination. HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE 2 (HSI2) of Arabidopsis thaliana is a B3 DNA-binding domain protein that represses the transcription of sugar-inducible reporter gene. Although disruption of HSI2 or HSI2-Like 1 (HSL1) did not affect growth, seeds with disruption of both HSI2 and HSL1 (KK mutant) developed abortive seedlings that stopped growing 7–9 days after imbibition. KK seedlings developed swollen hypocotyls that accumulated seed storage proteins and oil on medium containing sucrose or other metabolizable sugars, and calluses developed from KK seedlings also accumulated seed storage reserves. The expression of seed maturation genes, which include LEAFY COTYLEDON-type master regulators, in KK seedlings depended on the concentration of sucrose, suggesting that sugar controls the expression of seed maturation genes. Our results suggest that HSI2 and HSL1 repress the sugar-inducible expression of the seed maturation program in seedlings and play an essential role in regulating the transition from seed maturation to seedling growth.

Cell Identity Regulators Link Development and Stress Responses in the Arabidopsis Root

Stress responses in plants are tightly coordinated with developmental processes, but interaction of these pathways is poorly understood. We used genome-wide assays at high spatiotemporal resolution to understand the processes that link development and stress in the Arabidopsis root. Our meta-analysis finds little evidence for a universal stress response. However, common stress responses appear to exist with many showing cell type specificity. Common stress responses may be mediated by cell identity regulators because mutations in these genes resulted in altered responses to stress. Evidence for a direct role for cell identity regulators came from genome-wide binding profiling of the key regulator SCARECROW, which showed binding to regulatory regions of stress-responsive genes. Coexpression in response to stress was used to identify genes involved in specific developmental processes. These results reveal surprising linkages between stress and development at cellular resolution, and show the power of multiple genome-wide data sets to elucidate biological processes.

Analysis of a Sugar Response Mutant of Arabidopsis Identified a Novel B3 Domain Protein That Functions as an Active Transcriptional Repressor

A recessive mutation hsi2 of Arabidopsis (Arabidopsis thaliana) expressing luciferase (LUC) under control of a short promoter derived from a sweet potato (Ipomoea batatas) sporamin gene (Spomin∷LUC) caused enhanced LUC expression under both low- and high-sugar conditions, which was not due to increased level of abscisic acid. The hsi2 mutant contained a nonsense mutation in a gene encoding a protein with B3 DNA-binding domain. HSI2 and two other Arabidopsis proteins appear to constitute a novel subfamily of B3 domain proteins distinct from ABI3, FUS3, and LEC2, which are transcription activators involved in seed development. The C-terminal part of HSI2 subfamily proteins contained a sequence similar to the ERF-associated amphiphilic repression (EAR) motif. Deletion of the C-terminal portion of HSI2 lost in the hsi2 mutant caused reduced nuclear targeting of HSI2. Null allele of HSI2 showed even higher Spomin∷LUC expression than the hsi2 mutant, whereas overexpression of HSI2 reduced the LUC expression. Transient coexpression of 35S∷HSI2 with Spomin∷LUC in protoplasts repressed the expression of LUC activity, and deletion or mutation of the EAR motif significantly reduced the repression activity of HSI2. These results indicate that HSI2 and related proteins are B3 domain-EAR motif active transcription repressors.

An Oncoprotein from the Plant Pathogen Agrobacterium Has Histone Chaperone-Like Activity

Protein 6b, encoded by T-DNA from the pathogen Agrobacterium tumefaciens, stimulates the plant hormone–independent division of cells in culture in vitro and induces aberrant cell growth and the ectopic expression of various genes, including genes related to cell division and meristem-related class 1 KNOX homeobox genes, in 6b-expressing transgenic Arabidopsis thaliana and Nicotiana tabacum plants. Protein 6b is found in nuclei and binds to several plant nuclear proteins. Here, we report that 6b binds specifically to histone H3 in vitro but not to other core histones. Analysis by bimolecular fluorescence complementation revealed an interaction in vivo between 6b and histone H3. We recovered 6b from a chromatin fraction from 6b-expressing plant cells. A supercoiling assay and digestion with micrococcal nuclease indicated that 6b acts as a histone chaperone with the ability to mediate formation of nucleosomes in vitro. Mutant 6b, lacking the C-terminal region that is required for cell division–stimulating activity and interaction with histone H3, was deficient in histone chaperone activity. Our results suggest a relationship between alterations in nucleosome structure and the expression of growth-regulating genes on the one hand and the induction of aberrant cell proliferation on the other.

Defective root growth triggered by oxidative stress is controlled through the expression of cell cycle-related genes.

Reactive oxygen species (ROS) have many functions in aerobic organisms. High levels of ROS can have a negative impact on plant cells leading to senescence and cell death. ROS accumulates in cells subjected to environmental stress and induces a cellular response to this external stimulus. To protect cells from the negative impacts of excess ROS, plants also possess a ROS detoxifying system to maintain normal ROS levels. The regulation of ROS levels is particularly important as ROS also functions as an important signal molecule and can regulate plant growth by modulating gene expression. Despite the functional importance of ROS signaling, little is known about the molecular mechanisms involved in the regulation of gene expression through ROS. Therefore, the present study investigated the effect of hydrogen peroxide (H(2)O(2)), a ROS compound, on cell cycle-related gene expression. Gene expression analyses coupled with microdissected sections of the developmental zone of Arabidopsis root tips revealed that H(2)O(2) affects the expression of cell cycle-related genes. Additionally, ROS scavenging enzymes were found to play an important role in the root growth phenotype induced by H(2)O(2). Specifically, root growth inhibition by H(2)O(2) was diminished in transgenic Arabidopis overexpressing peroxidase but increased in a catalase2 (cat2) mutant. The strong root growth inhibition observed in the cat2 mutant upon H(2)O(2) treatment indicated that CAT2 has an essential role in maintaining root meristem activity in the presence of oxidative stress. Overall, these results confirm that ROS function not only as stress-related compounds but that they also function as signaling molecules to regulate the progression of the cell cycle in root tips.

Wound-induced expression of DEFECTIVE IN ANTHER DEHISCENCE1 and DAD1-like lipase genes is mediated by both CORONATINE INSENSITIVE1-dependent and independent pathways in Arabidopsis thaliana.

Key messageEndogenous JA production is not necessary for wound-induced expression of JA-biosynthetic lipase genes such asDAD1in Arabidopsis. However, the JA-Ile receptor COI1 is often required for their JA-independent induction.AbstractWounding is a serious event in plants that may result from insect feeding and increase the risk of pathogen infection. Wounded plants produce high amounts of jasmonic acid (JA), which triggers the expression of insect and pathogen resistance genes. We focused on the transcriptional regulation of DEFECTIVE IN ANTHER DEHISCENCE1 and six of its homologs including DONGLE (DGL) in Arabidopsis, which encode lipases involved in JA biosynthesis. Plants constitutively expressing DAD1 accumulated a higher amount of JA than control plants after wounding, indicating that the expression of these lipase genes contributes to determining JA levels. We found that the expression of DAD1, DGL, and other DAD1-LIKE LIPASE (DALL) genes is induced upon wounding. Some DALLs were also expressed in unwounded leaves. Further experiments using JA-biosynthetic and JA-response mutants revealed that the wound induction of these genes is regulated by several distinct pathways. DAD1 and most of its homologs other than DALL4 were fully induced without relying on endogenous JA-Ile production and were only partly affected by JA deficiency, indicating that positive feedback by JA is not necessary for induction of these genes. However, DAD1 and DGL required CORONATINE INSENSITIVE1 (COI1) for their expression, suggesting that a molecule other than JA might act as a regulator of COI1. Wound induction of DALL1, DALL2, and DALL3 did not require COI1. This differential regulation of DAD1 and its homologs might explain their functions at different time points after wounding.

MYB30 links ROS signaling, root cell elongation, and plant immune responses

Significance Plant roots tune their growth to the environment. An important class of molecules involved in environmental responses as well as in root growth regulation is composed of reactive oxygen species (ROS). By making use of a comprehensive transcriptome atlas capturing ROS responses in different developmental zones of the root, we uncovered a regulatory network that is involved in root-growth regulation and responses to biotic stress. This network is composed of the ROS-responsive transcription factor MYB30, which regulates multiple genes involved in the transport of very-long-chain fatty acids (VLCFAs). Overall, our findings show that Arabidopsis uses the same MYB30-dependent regulatory network for root-growth and immunity responses, processes that were considered largely independent of each other. Reactive oxygen species (ROS) are known to be important signal molecules that are involved in biotic and abiotic stress responses as well as in growth regulation. However, the molecular mechanisms by which ROS act as a growth regulator, as well as how ROS-dependent growth regulation relates to its roles in stress responses, are not well understood. We performed a time-course microarray analysis of Arabidopsis root tips upon treatment with hydrogen peroxide, which we named “ROS-map.” Using the ROS-map, we identified an MYB transcription factor, MYB30, which showed a strong response to ROS treatment and is the key regulator of a gene network that leads to the hydrogen peroxide-dependent inhibition of root cell elongation. Intriguingly, this network contained multiple genes involved in very-long-chain fatty acid (VLCFA) transport. Finally, we showed that MYB30 is necessary for root growth regulation during defense responses, thus providing a molecular link between these two ROS-associated processes.

Development rooted in interwoven networks

Freshwater planarians appear to utilize inductive signals to specify their germ cell lineage: germ cells are believed to form post-embryonically from the pluripotent somatic stem cells, known as neoblasts. Previously, we identified a planarian homolog of nanos (Smed-nanos) and demonstrated by RNA interference (RNAi) that this gene is required for the development, maintenance, and regeneration of planarian germ cells. We have performed microarray analyses to compare gene expression profiles between planarians with early germ cells and those without them. We identified ∼300 genes that are significantly down-regulated in animals lacking early germ cells. This data set contains genes implicated in germ cell development in other organisms, conserved genes not yet reported to have germ cell-related functions, and novel genes. Analysis using putative domain functions (Clusters of Orthologous Groups) suggested diverse molecular functions, including cytoskeletal components, metabolism, RNA processing and modification, transcription, as well as signal transduction. Top hits have been validated by in situ hybridization. Functional analyses of these genes via RNA interference are being carried out. Thus far, we have identified several genes that, when knocked down by RNAi, cause various defects in germ cell development, including: impaired testes development; loss of spermatogonial stem cells; meiotic failure; and defects in sperm elongation. This work will contribute to our knowledge of conserved regulators of germ cell differentiation. (Supported by NIH-NICHD R01-HD043403.)