Nuclear growth and import can be uncoupled.

What drives nuclear growth? Studying nuclei assembled in Xenopus egg extract and focusing on importin α/ß-mediated nuclear import, we show that, while import is required for nuclear growth, nuclear growth and import can be uncoupled when chromatin structure is manipulated. Nuclei treated with micrococcal nuclease to fragment DNA grew slowly despite exhibiting little to no change in import rates. Nuclei assembled around axolotl chromatin with 20-fold more DNA than Xenopus grew larger but imported more slowly. Treating nuclei with reagents known to alter histone methylation or acetylation caused nuclei to grow less while still importing to a similar extent or to grow larger without significantly increasing import. Nuclear growth but not import was increased in live sea urchin embryos treated with the DNA methylator N-nitrosodimethylamine. These data suggest that nuclear import is not the primary driving force for nuclear growth. Instead, we observed that nuclear blebs expanded preferentially at sites of high chromatin density and lamin addition, whereas small Benzonase-treated nuclei lacking DNA exhibited reduced lamin incorporation into the nuclear envelope. In summary, we report experimental conditions where nuclear import is not sufficient to drive nuclear growth, hypothesizing that this uncoupling is a result of altered chromatin structure.

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.

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.