AtHMGB15 regulates tapetal apoptosis in pollen development and actin dynamics during pollen germination in arabidopsis.

KEY MESSAGE: ARID-HMG DNA binding protein, AtHMGB15, regulates pollen development and pollen germination in Arabidopsis. Previous studies have shown that ARID-HMG DNA binding protein, AtHMGB15 regulate pollen development and pollen germination in Arabidopsis. Here, we performed transcriptome and cytological studies to understand the role of AtHMGB15 in regulating pollen wall morphology and the pollen tube germination rate. Our result showed abnormal vacuolization in the tapetal cells during anther maturation and prolonged PCD in AtHMGB15 loss-of-function mutant. The tapetum has the ability to perform both secretory and biosynthetic activities critical for pollen maturation and pollen viability. Interestingly, expression of PCD executer genes CEP1, MC9 and RNS3 were significant down-regulation of in athmgb15-4. The growth of pollen tubes is regulated by the actin cytoskeleton dynamics. To address the defect in pollen tube growth of athmgb15, we monitored the actin network in growing pollen tubes of wildtype and athmgb15-4 using Rhodamine-phalloidin fluorescence. Our results indicate a highly fragmented actin distribution in athmgb15-4 pollen tubes with a lesser number of long actin fibers and significantly low f-actin concentration at the apex. q-RTPCR further indicates significant downy-regulation of actin regulatory proteins VLN2 and PRF4. Collectively, our results suggest that AtHMGB15 being a nuclear architectural protein orchestrates high-order chromatin organization to promote the transcription of genes responsible for pollen development and pollen germination.

The Arabidopsis ARID-HMG DNA-BINDING PROTEIN 15 modulates JA signaling by regulating MYC2 during pollen development.

The intricate process of male gametophyte development in flowering plants is regulated by jasmonic acid (JA) signaling. JA signaling initiates with the activation of the basic-helix-loop-helix (bHLH) transcription factor (TF), MYC2, leading to the expression of numerous JA-responsive genes during stamen development and pollen maturation. However, the regulation of JA signaling during different stages of male gametophyte development remains less understood. This study focuses on the characterization of the plant ARID-HMG DNA-BINDING PROTEIN 15 (AtHMGB15), and its role in pollen development in Arabidopsis (Arabidopsis thaliana). Phenotypic characterization of a T-DNA insertion line (athmgb15-4) revealed delayed bolting, shorter siliques, and reduced seed set in mutant plants compared to the wildtype. Additionally, AtHMGB15 deletion resulted in defective pollen morphology, delayed pollen germination, aberrant pollen tube growth, and a higher percentage of non-viable pollen grains. Molecular analysis indicated the down-regulation of JA biosynthesis and signaling genes in the athmgb15-4 mutant. Quantitative analysis demonstrated that jasmonic acid and its derivatives were approximately tenfold lower in athmgb15-4 flowers. Exogenous application of methyl jasmonate could restore pollen morphology and germination, suggesting that the low JA content in athmgb15-4 impaired JA signaling during pollen development. Furthermore, our study revealed that AtHMGB15 physically interacts with MYC2 to form a transcription activation complex. This complex promotes the transcription of key JA signaling genes, the R2R3-MYB TFs MYB21 and MYB24, during stamen and pollen development. Collectively, our findings highlight the role of AtHMGB15 as a positive regulator of the JA pathway, controlling the spatiotemporal expression of key regulators involved in Arabidopsis stamen and pollen development.

Identification of genome-wide targets and DNA recognition sequence of the Arabidopsis HMG-box protein AtHMGB15 during cold stress response.

AtHMGB15 belongs to a group of ARID-HMG proteins which are plant specific. The presence of two known DNA binding domains: AT rich interacting domain (ARID) and High Mobility Group (HMG)-box, in one polypeptide, makes this protein intriguing. Although proteins containing individual HMG and ARID domains have been characterized, not much is known about the role of ARID-HMG proteins. Promoter analysis of AtHMGB15 showed the presence of various stress responsive cis regulatory elements along with MADS-box containing transcription factors. Our result shows that the expression of AtHMGB15 increased significantly upon application of cold stress. Using ChIP-chip approach, we have identified 6128 and 4689 significantly enriched loci having AtHMGB15 occupancy under control and cold stressed condition respectively. GO analysis shows genes belonging to abiotic stress response, cold response and root development were AtHMGB15 targets during cold stress. DNA binding and footprinting assays further identified A(A/C)--ATA---(A/T)(A/T) as AtHMGB15 binding motif. The enriched probe distribution in both control and cold condition shows a bias of AtHMGB15 binding towards the transcribed (gene body) region. Further, the expression of cold stress responsive genes decreased in athmgb15 knockout plants compared to wild-type. Taken together, binding enrichment of AtHMGB15 to the promoter and upstream to stress loci suggest an unexplored role of the protein in stress induced transcription regulation.