ABI3 promotes auxin signalling by regulating SHY2 expression to control primary root growth in response to dehydration stress.

Plants combat dehydration stress through several adaptive measures including root architectural changes. Here we show that when exposed to varying levels of dehydration stress, primary root growth in Arabidopsis is modulated by regulating root meristem activity. ABA in concert with auxin signalling perceives the stress level and adapts primary root growth accordingly. ABI3, the ABA responsive transcription factor stands at the intersection of ABA and auxin signalling and fine tunes primary root growth in response to dehydration stress. Under low ABA or dehydration stress, induction of ABI3 expression promotes auxin signalling by decreasing expression of SHY2, a negative regulator of auxin response. This further enhances the expression of auxin transporter gene PIN1 and cell cycle gene CYCB1;1, resulting in an increase in primary root meristem size and root length. Higher levels of dehydration stress or ABA repress ABI3 expression and promote ABI5 expression. This elevates SHY2 expression, thereby impairing primary root meristem activity and retarding root growth. Notably, ABI5 can promote SHY2 expression only in the absence of ABI3. Such ABA concentration dependent expression of ABI3 therefore functions as a regulatory sensor of dehydration stress levels and orchestrates primary root growth by coordinating its downstream regulon.

RNA Polymerase II Dependent Crosstalk between H4K16 Deacetylation and H3K56 Acetylation Promotes Transcription of Constitutively Expressed Genes.

Nucleosome dynamics in the coding region of a transcriptionally active locus is critical for understanding how RNA polymerase II progresses through the gene body. Histone acetylation and deacetylation critically influence nucleosome accessibility during DNA metabolic processes like transcription. Effect of such histone modifications is context and residue dependent. Rather than effect of individual histone residues, the network of modifications of several histone residues in combination generates a chromatin landscape that is conducive for transcription. Here we show that in Saccharomyces cerevisiae, crosstalk between deacetylation of the H4 N-terminal tail residue H4K16 and acetylation of the H3 core domain residue H3K56, promotes RNA polymerase II progression through the gene body. Results indicate that deacetylation of H4K16 precedes and in turn induces H3K56 acetylation. Effectively, recruitment of Rtt109, the HAT responsible for H3K56 acetylation is essentially dependent on H4K16 deacetylation. In Hos2 deletion strains, where H4K16 deacetylation is abolished, both H3K56 acetylation and RNA polymerase II recruitment gets significantly impaired. Notably, H4K16 deacetylation and H3K56 acetylation are found to be essentially dependent on active transcription. In summary, H4K16 deacetylation promotes H3K56 acetylation and the two modifications together work towards successful functioning of RNA polymerase II during active transcription.

RAV1 mediates cytokinin signaling for regulating primary root growth in Arabidopsis.

Root growth dynamics is an outcome of complex hormonal crosstalk. The primary root meristem size, for example, is determined by antagonizing actions of cytokinin and auxin. Here we show that RAV1, a member of the AP2/ERF family of transcription factors, mediates cytokinin signaling in roots to regulate meristem size. The rav1 mutants have prominently longer primary roots, with a meristem that is significantly enlarged and contains higher cell numbers, compared with wild-type. The mutant phenotype could be restored on exogenous cytokinin application or by inhibiting auxin transport. At the transcript level, primary cytokinin-responsive genes like ARR1, ARR12 were significantly downregulated in the mutant root, indicating impaired cytokinin signaling. In concurrence, cytokinin induced regulation of SHY2, an Aux/IAA gene, and auxin efflux carrier PIN1 was hindered in rav1, leading to altered auxin transport and distribution. This effectively altered root meristem size in the mutant. Notably, CRF1, another member of the AP2/ERF family implicated in cytokinin signaling, is transcriptionally repressed by RAV1 to promote cytokinin response in roots. Further associating RAV1 with cytokinin signaling, our results demonstrate that cytokinin upregulates RAV1 expression through ARR1, during post-embryonic root development. Regulation of RAV1 expression is a part of secondary cytokinin response that eventually represses CRF1 to augment cytokinin signaling. To conclude, RAV1 functions in a branch pathway downstream to ARR1 that regulates CRF1 expression to enhance cytokinin action during primary root development in Arabidopsis.

DNA methylation and regulation of gene expression: Guardian of our health.

One of the most critical epigenetic signatures present in the genome of higher eukaryotes is the methylation of DNA at the C-5 position of the cytosine ring. Based on the sites of DNA methylation in a locus, it can serve as a repressive or activation mark for gene expression. In a crosstalk with histone modifiers, DNA methylation can consequently either inhibit binding of the transcription machinery or generate a landscape conducive for transcription. During developmental phases, the DNA methylation pattern in the genome undergoes alterations as a result of regulated balance between de novo DNA methylation and demethylation. Resultantly, differentiated cells inherit a unique DNA methylation pattern that fine tunes tissue-specific gene expression. Although apparently a stable epigenetic mark, DNA methylation is actually labile and is a complex reflection of interaction between epigenome, genome and environmental factors prior to birth and during progression of life. Recent findings indicate that levels of DNA methylation in an individual is a dynamic outcome, strongly influenced by the dietary environment during germ cell formation, embryogenesis and post birth exposures. Loss of balances in DNA methylation during developmental stages may result in imprinting disorders, while at any later stage may lead to increased predisposition to various diseases and abnormalities. This review aims to provide an outline of how our epigenome is uniquely guided by our lifetime of experiences beginning in the womb and how understanding it better holds future possibilities of improvised clinical applications.

Deacetylation of H4 lysine16 affects acetylation of lysine residues in histone H3 and H4 and promotes transcription of constitutive genes.

Histone modification map of H4 N-terminal tail residues in Saccharomyces cerevisiae reveals the prominence of lysine acetylation. Previous reports have indicated the importance of lysine acetylation in maintaining chromatin structure and function. H4K16, a residue with highly regulated acetylation dynamics has unique functions not overlapping with the other H4 N- terminal acetylable residues. The present work unravels the role of H4K16 acetylation in regulating expression of constitutive genes. H4K16 gets distinctly deacetylated over the coding region of constitutively expressed genes. Deacetylation of H4K16 reduces H3K9 acetylation at the cellular and gene level. Reduced H3K9 acetylation however did not negatively correlate with active gene transcription. Significantly, H4K16 deacetylation was found to be associated with hypoacetylated H4K12 throughout the locus of constitutive genes. H4K16 and K12 deacetylation is known to favour active transcription. Sas2, the HAT mutant showed similar patterns of hypoacetylated H3K9 and H4K12 at the active loci, clearly implying that the modifications were associated with deacetylation state of H4K16. Deacetylation of H4K16 was also concurrent with increased H3K56 acetylation in the promoter region and ORF of the constitutive genes. Combination of all these histone modifications significantly reduced H3 occupancy, increased promoter accessibility and enhanced RNAPII recruitment at the constitutively active loci. Consequently, we found that expression of active genes was higher in H4K16R mutant which mimic deacetylated state, but not in H4K16Q mimicking constitutive acetylation. To summarize, H4K16 deacetylation linked with H4K12 and H3K9 hypoacetylation along with H3K56 hyperacetylation generate a chromatin landscape that is conducive for transcription of constitutive genes.

ABI3 plays a role in de-novo root regeneration from Arabidopsis thaliana callus cells.

Developmental plasticity and the ability to regenerate organs during the life cycle are a signature feature of plant system. De novo organogenesis is a common mode of plant regeneration and may occur directly from the explant or indirectly via callus formation. It is now evident that callus formation occurs through the root development pathway. In fact, callus cells behave like a group of root primordium cells that are under the control of exogenous auxin. Presence or absence of auxin decides the subsequent fate of these cells. While in presence of external supplementation of auxin they are maintained as root primordia cells, absence of exogenous auxin induces the callus cells into patterning, differentiation and finally root emergence. Here we show that in absence of functional ABI3, a prominent member of the B3 superfamily of transcription factors, root regeneration is compromised in Arabidopsis callus cells. In culture medium free of any exogenous hormone supplementation, while adventitious root emergence and growth was prominently observed in wild type cells, no such features were observed in abi3-6 cells. Expression of auxin-responsive AUX1 and GH3 genes was significantly reduced in abi3-6 cells, indicating that auxin levels or distribution may be altered in absence of ABI3.

ABI3 mediated repression of RAV1 gene expression promotes efficient dehydration stress response in Arabidopsis thaliana.

Dehydration stress response is a complex mechanism in plants involving several factors and hormone signalling pathways. RAV1 is a member of the AP2/ERF family of transcription factors that works in various developmental pathways. Here we show that downregulation of RAV1 gene expression is important for efficient dehydration stress response. Interestingly, the B3-domain transcription factor ABI3 negatively regulates RAV1 expression. In absence of ABI3, RAV1 expression increases during dehydration stress compared to control. As a part of stress response, ABI3 occupancy increases in the RAV1 promoter region. Such regulation of RAV1 gene expression seems vital as absence of RAV1 leads to reduced water loss during dehydration stress and consequently faster recovery compared to wild type. rav1 mutant seedlings show more abundant root growth under control condition and higher primary root elongation compared to wild type when subjected to dehydration stress. Mutants also exhibit enhanced ABA sensitivity compared to wild type. At the transcript level, rooting genes like NAC1, ARF16, SLR and SLR-downstream genes like ARF7, PLT3, SHR show differential expression in rav1 mutant, compared to wild type. Additionally, ethylene-responsive genes ETR1, EIN2 and ERF1 also get differentially expressed in presence and absence of RAV1 under control and stress conditions. This indicates an altered ethylene response in the rav1 mutant. All these features render rav1seedlings better equipped for responding to dehydration stress. It thus becomes evident that ABI3 mediated regulation of RAV1 gene expression is a significant part of dehydration stress signalling for efficient stress management at the molecular and morphological level.

Regulated acetylation and deacetylation of H4 K16 is essential for efficient NER in Saccharomyces cerevisiae.

Acetylation status of H4 K16, a residue in the histone H4 N-terminal tail plays a unique role in regulating chromatin structure and function. Here we show that, during UV-induced nucleotide excision repair H4 K16 gets hyperacetylated following an initial phase of hypoacetylation. Disrupting H4 K16 acetylation-deacetylation by mutating H4 K16 to R (deacetylated state) or Q (acetylated state) leads to compromised chromatin functions. In the silenced mating locus and telomere region H4 K16 mutants show higher recruitment of Sir proteins and spreading beyond the designated boundaries. More significantly, chromatin of both the H4 K16 mutants has reduced accessibility in the silenced regions and genome wide. On UV irradiation, the mutants showed higher UV sensitivity, reduced NER rate and altered H3 N-terminal tail acetylation, compared to wild type. NER efficiency is affected by reduced or delayed recruitment of early NER proteins and chromatin remodeller Swi/Snf along with lack of nucleosome rearrangement during repair. Additionally UV-induced expression of RAD and SNF5 genes was reduced in the mutants. Hindered chromatin accessibility in the H4 K16 mutants is thus non-conducive for gene expression as well as recruitment of NER and chromatin remodeller proteins. Subsequently, inadequate nucleosomal rearrangement during early phases of repair impeded accessibility of the NER complex to DNA lesions, in the H4 K16 mutants. Effectively, NER efficiency was found to be compromised in the mutants. Interestingly, in the transcriptionally active chromatin region, both the H4 K16 mutants showed reduced NER rate during early repair time points. However, with progression of repair H4 K16R repaired faster than K16Q mutants and rate of CPD removal became differential between the two mutants during later NER phases. To summarize, our results establish the essentiality of regulated acetylation and deacetylation of H4 K16 residue in maintaining chromatin accessibility and efficiency of functions like NER and gene expression.

Transcription factor ABI3 auto-activates its own expression during dehydration stress response.

The transcription factor abscisic acid insensitive 3 (ABI3) has recently been shown to mediate the dehydration stress response in nonseed and seed plants by regulation of several downstream genes. Here, we show how ABI3 autoregulates its transcription in response to dehydration stress signalling. Autoactivation is primarily through the Sph/RY element CATGCA present at the promoter region of ABI3. Along with other known cis-elements found at the ABI3 promoter, CATGCA remains occluded by nucleosomes during transcription repression. The nucleosomes tend to reposit during active transcription and are associated with several histone modifications, such as H3K9 and K27 acetylations and H3K4 trimethylation. This work thus, reveals the genetic and epigenetic essentials required for expression of the ABI3 gene, a crucial factor regulating dehydration stress signalling in Arabidopsis thaliana.

ABI3 mediates dehydration stress recovery response in Arabidopsis thaliana by regulating expression of downstream genes.

ABI3, originally discovered as a seed-specific transcription factor is now implicated to act beyond seed physiology, especially during abiotic stress. In non-seed plants, ABI3 is known to act in desiccation stress signaling. Here we show that ABI3 plays a role in dehydration stress response in Arabidopsis. ABI3 gene was upregulated during dehydration stress and its expression was maintained during subsequent stress recovery phases. Comparative gene expression studies in response to dehydration stress and stress recovery were done with genes which had potential ABI3 binding sites in their upstream regulatory regions. Such studies showed that several genes including known seed-specific factors like CRUCIFERIN1, CRUCIFERIN3 and LEA-group of genes like LEA76, LEA6, DEHYDRIN LEA and LEA-LIKE got upregulated in an ABI3-dependent manner, especially during the stress recovery phase. ABI3 got recruited to regions upstream to the transcription start site of these genes during dehydration stress response through direct or indirect DNA binding. Interestingly, ABI3 also binds to its own promoter region during such stress signaling. Nucleosomes covering potential ABI3 binding sites in the upstream sequences of the above-mentioned genes alter positions, and show increased H3 K9 acetylation during stress-induced transcription. ABI3 thus mediates dehydration stress signaling in Arabidopsis through regulation of a group of genes that play a role primarily during stress recovery phase.