ACBP4-WRKY70-RAP2.12 module positively regulates submergence-induced hypoxia response in Arabidopsis thaliana.

ACYL-CoA-BINDING PROTEINs (ACBPs) play crucial regulatory roles during plant response to hypoxia, but their molecular mechanisms remain poorly understood. Our study reveals that ACBP4 serves as a positive regulator of the plant hypoxia response by interacting with WRKY70, influencing its nucleocytoplasmic shuttling in Arabidopsis thaliana. Furthermore, we demonstrate the direct binding of WRKY70 to the ACBP4 promoter, resulting in its upregulation and suggesting a positive feedback loop. Additionally, we pinpointed a phosphorylation site at Ser638 of ACBP4, which enhances submergence tolerance, potentially by facilitating WRKY70's nuclear shuttling. Surprisingly, a natural variation in this phosphorylation site of ACBP4 allowed A. thaliana to adapt to humid conditions during its historical demographic expansion. We further observed that both phosphorylated ACBP4 and oleoyl-CoA can impede the interaction between ACBP4 and WRKY70, thus promoting WRKY70's nuclear translocation. Finally, we found that the overexpression of orthologous BnaC5.ACBP4 and BnaA7.WRKY70 in Brassica napus increases submergence tolerance, indicating their functional similarity across genera. In summary, our research not only sheds light on the functional significance of the ACBP4 gene in hypoxia response, but also underscores its potential utility in breeding flooding-tolerant oilseed rape varieties.

Coordination between two cis-elements of WRKY33, bound by the same transcription factor, confers humid adaption in Arabidopsis thaliana.

To cope with flooding-induced hypoxia, plants have evolved different strategies. Molecular strategies, such as the N-degron pathway and transcriptional regulation, are known to be crucial for Arabidopsis thaliana's hypoxia response. Our study uncovered a novel molecular strategy that involves a single transcription factor interacting with two identical cis-elements, one located in the promoter region and the other within the intron. This unique double-element adjustment mechanism has seldom been reported in previous studies. In humid areas, WRKY70 plays a crucial role in A. thaliana's adaptation to submergence-induced hypoxia by binding to identical cis-elements in both the promoter and intron regions of WRKY33. This dual binding enhances WRKY33 expression and the activation of hypoxia-related genes. Conversely, in arid regions lacking the promoter cis-element, WRKY70 only binds to the intron cis-element, resulting in limited WRKY33 expression during submergence stress. The presence of a critical promoter cis-element in humid accessions, but not in dry accessions, indicates a coordinated regulation enabling A. thaliana to adapt and thrive in humid habitats.

Scattered differentiation of unlinked loci across the genome underlines ecological divergence of the selfing grass Brachypodium stacei.

Ecological divergence without geographic isolation, as an early speciation process that may lead finally to reproductive isolation through natural selection, remains a captivating topic in evolutionary biology. However, the pattern of genetic divergence underlying this process across the genome may vary between species and mating systems. Here, we present evidence that Brachypodium stacei, an annual and highly selfing grass model species, has undergone sympatric ecological divergence without geographic isolation. Genomic, transcriptomic, and metabolomic analyses together with lab experiments mimicking the two opposite environmental conditions suggest that diploid B. stacei populations have diverged sympatrically in two slopes characterized by distinct biomes at Evolution Canyon I (ECI), Mount Carmel, Israel. Despite ongoing gene flow, primarily facilitated by seed dispersal, the level of gene flow has progressively decreased over time. This local adaptation involves the scattered divergence of many unlinked loci across the total genome that include both coding genes and noncoding regions. Additionally, we have identified significant differential expressions of genes related to the ABA signaling pathway and contrasting metabolome composition between the arid- vs. forest-adapted B. stacei populations in ECI. These results suggest that multiple small loci involved in environmental responses act additively to account for ecological adaptations by this selfing species in contrasting environments.

The pan-genome and local adaptation of Arabidopsis thaliana.

Arabidopsis thaliana serves as a model species for investigating various aspects of plant biology. However, the contribution of genomic structural variations (SVs) and their associate genes to the local adaptation of this widely distribute species remains unclear. Here, we de novo assemble chromosome-level genomes of 32 A. thaliana ecotypes and determine that variable genes expand the gene pool in different ecotypes and thus assist local adaptation. We develop a graph-based pan-genome and identify 61,332 SVs that overlap with 18,883 genes, some of which are highly involved in ecological adaptation of this species. For instance, we observe a specific 332 bp insertion in the promoter region of the HPCA1 gene in the Tibet-0 ecotype that enhances gene expression, thereby promotes adaptation to alpine environments. These findings augment our understanding of the molecular mechanisms underlying the local adaptation of A. thaliana across diverse habitats.

Natural variation in the SVP contributes to the pleiotropic adaption of Arabidopsis thaliana across contrasted habitats.

Coordinated plant adaptation involves the interplay of multiple traits driven by habitat-specific selection pressures. Pleiotropic effects, wherein genetic variants of a single gene control multiple traits, can expedite such adaptations. Until present, only a limited number of genes have been reported to exhibit pleiotropy. Here, we create a recombinant inbred line (RIL) population derived from two Arabidopsis thaliana (A. thaliana) ecotypes originating from divergent habitats. Using this RIL population, we identify an allelic variation in a MADS-box transcription factor, SHORT VEGETATIVE PHASE (SVP), which exerts a pleiotropic effect on leaf size and drought-versus-humidity tolerance. Further investigation reveals that a natural null variant of the SVP protein disrupts its normal regulatory interactions with target genes, including GRF3, CYP707A1/3, and AtBG1, leading to increased leaf size, enhanced tolerance to humid conditions, and changes in flowering time of humid conditions in A. thaliana. Remarkably, polymorphic variations in this gene have been traced back to early A. thaliana populations, providing a genetic foundation and plasticity for subsequent colonization of diverse habitats by influencing multiple traits. These findings advance our understanding of how plants rapidly adapt to changing environments by virtue of the pleiotropic effects of individual genes on multiple trait alterations.

Allelic shift in cis-elements of the transcription factor RAP2.12 underlies adaptation associated with humidity in Arabidopsis thaliana.

Populations of widespread species are usually geographically distributed through contrasting stresses, but underlying genetic mechanisms controlling this adaptation remain largely unknown. Here, we show that in Arabidopsis thaliana, allelic changes in the cis-regulatory elements, WT box and W box, in the promoter of a key transcription factor associated with oxygen sensing, RELATED TO AP 2.12 (RAP2.12), are responsible for differentially regulating tolerance to drought and flooding. These two cis-elements are regulated by different transcription factors that downstream of RAP2.12 results in differential accumulation of hypoxia-responsive transcripts. The evolution from one cis-element haplotype to the other is associated with the colonization of humid environments from arid habitats. This gene thus promotes both drought and flooding adaptation via an adaptive mechanism that diversifies its regulation through noncoding alleles.

The U-box E3 ubiquitin ligase PalPUB79 positively regulates ABA-dependent drought tolerance via ubiquitination of PalWRKY77 in Populus.

The abscisic acid (ABA) signalling pathway is involved in the plant response to osmotic stress caused by drought and/or salinity. Although the ABA signalling pathway has been elucidated in Arabidopsis, it remains elusive in woody poplars. In this study, genome-wide analyses of U-box genes in poplars revealed that a U-box E3 ubiquitin ligase gene, PalPUB79, is significantly induced following drought, salinity and ABA signalling. PalPUB79 overexpression enhanced drought tolerance in transgenic poplars, while PalPUB79 RNAi lines were more sensitive to drought. PalPUB79 positively regulated ABA signalling pathway. Furthermore, PalPUB79 interacted with PalWRKY77, a negative transcriptional regulator of ABA signalling, and mediated its ubiquitination for degradation, therefore counteracting its inhibitory effect on PalRD26 transcription. However, the finding that PalWRKY77 negatively regulates PalPUB79 expression was indicative of a negative feedback loop between PalWRKY77 and PalPUB79 during ABA signalling in poplar. These findings provide novel insight into the mechanism through which PalPUB79 enhances the ABA-mediated stress response in woody poplars.

A chromosome-level Camptotheca acuminata genome assembly provides insights into the evolutionary origin of camptothecin biosynthesis.

Camptothecin and its derivatives are widely used for treating malignant tumors. Previous studies revealed only a limited number of candidate genes for camptothecin biosynthesis in Camptotheca acuminata, and it is still poorly understood how its biosynthesis of camptothecin has evolved. Here, we report a high-quality, chromosome-level C. acuminata genome assembly. We find that C. acuminata experiences an independent whole-genome duplication and numerous genes derive from it are related to camptothecin biosynthesis. Comparing with Catharanthus roseus, the loganic acid O-methyltransferase (LAMT) in C. acuminata fails to convert loganic acid into loganin. Instead, two secologanic acid synthases (SLASs) convert loganic acid to secologanic acid. The functional divergence of the LAMT gene and positive evolution of two SLAS genes, therefore, both contribute greatly to the camptothecin biosynthesis in C. acuminata. Our results emphasize the importance of high-quality genome assembly in identifying genetic changes in the evolutionary origin of a secondary metabolite.

The ubiquitin E3 ligase SR1 modulates the submergence response by degrading phosphorylated WRKY33 in Arabidopsis.

Oxygen deprivation caused by flooding activates acclimation responses to stress and restricts plant growth. After experiencing flooding stress, plants must restore normal growth; however, which genes are dynamically and precisely controlled by flooding stress remains largely unknown. Here, we show that the Arabidopsis thaliana ubiquitin E3 ligase SUBMERGENCE RESISTANT1 (SR1) regulates the stability of the transcription factor WRKY33 to modulate the submergence response. SR1 physically interacts with WRKY33 in vivo and in vitro and controls its ubiquitination and proteasomal degradation. Both the sr1 mutant and WRKY33 overexpressors exhibited enhanced submergence tolerance and enhanced expression of hypoxia-responsive genes. Genetic experiments showed that WRKY33 functions downstream of SR1 during the submergence response. Submergence induced the phosphorylation of WRKY33, which enhanced the activation of RAP2.2, a positive regulator of hypoxia-response genes. Phosphorylated WRKY33 and RAP2.2 were degraded by SR1 and the N-degron pathway during reoxygenation, respectively. Taken together, our findings reveal that the on-and-off module SR1-WRKY33-RAP2.2 is connected to the well-known N-degron pathway to regulate acclimation to submergence in Arabidopsis. These two different but related modulation cascades precisely balance submergence acclimation with normal plant growth.

CHYR1 ubiquitinates the phosphorylated WRKY70 for degradation to balance immunity in Arabidopsis thaliana.

It is critically important for plants to control the trade-off between normal growth and pathogen immunity. However, the underlying molecular mechanism remains largely unknown. Here we report such a mechanism controlled by WRKY70 and its partner CHYR1 in Arabidopsis. We found that both levels of the WRKY70 target gene SARD1 and the phosphorylated forms of WRKY70 were increased in WRKY70OE plants upon Pst DC3000 infection. Mechanistically, phosphorylation of WRKY70 at Thr22 and Ser34 occurs, which then activates SARD1 expression through binding to a WT box. Phosphorylated WRKY70 is degraded by 26S proteasome via CHYR1 when resuming normal growth after infection. In addition, nonphosphorylated WRKY70 represses SARD1 expression by binding to both W (inhibitory activity site) and WT (active activity site) boxes. The binding of WRKY70 to alternative cis-elements of SARD1 through a phosphorylation-mediated switch controlled by CHYR1 contributes to modulating the balance between immunity and growth.

The PalWRKY77 transcription factor negatively regulates salt tolerance and abscisic acid signaling in Populus.

High salinity, one of the most widespread abiotic stresses, inhibits photosynthesis, reduces vegetation growth, blocks respiration and disrupts metabolism in plants. In order to survive their long-term lifecycle, trees, such as Populus species, recruit the abscisic acid (ABA) signaling pathway to adapt to a saline environment. However, the molecular mechanism behind the ABA-mediated salt stress response in woody plants remains elusive. We have isolated a WRKY transcription factor gene, PalWRKY77, from Populus alba var. pyramidalis (poplar), the expression of which is repressed by salt stress. PalWRKY77 decreases salt tolerance in poplar. Furthermore, PalWRKY77 negatively regulated ABA-responsive genes and relieved ABA-mediated growth inhibition, indicating that PalWRKY77 is a repressor of the ABA response. In vivo and in vitro assays revealed that PalWRKY77 targets the ABA- and salt-induced PalNAC002 and PalRD26 genes by binding to the W-boxes in their promoters. In addition, overexpression of both PalNAC002 and PalRD26 could elevate salt tolerance in transgenic poplars. These findings reveal a novel negative regulation mechanism for the ABA signaling pathway mediated by PalWRKY77 that results in more sensitivity to salt stress in poplar. This deepens our understanding of the complex responses of woody species to salt stress.

WRKY33 interacts with WRKY12 protein to up-regulate RAP2.2 during submergence induced hypoxia response in Arabidopsis thaliana.

Tolerance of hypoxia is essential for most plants, but the underlying mechanisms are largely unknown. Here we show that adaptation to submergence induced hypoxia in Arabidopsis involves up-regulation of RAP2.2 through interactive action of WRKY33 and WRKY12. WRKY33- or WRKY12-overexpressing plants showed enhanced resistance to hypoxia. Y2H, BiFC, Co-IP and pull-down experiments confirmed the interaction of WRKY33 with WRKY12. Genetic experiments showed that RAP2.2 acts downstream of WRKY33/WRKY12. WRKY33 and WRKY12 can bind to and activate RAP2.2 individually. Genetic and molecular experiments demonstrate that the two WRKYs can synergistically enhance activation towards RAP2.2 to increase hypoxia tolerance. WRKY33 expression is increased in RAP2.2-overexpressing plants, indicating a feedback regulation by RAP2.2 during submergence process, which was corroborated by EMSA, ChIP, dual-LUC and genetic experiments. Our results show that a regulatory cascade module involving WRKY33, WRKY12 and RAP2.2 plays a key role in submergence induced hypoxia response of Arabidopsis and illuminate functions of WRKYs in hypoxia tolerance.

The PalERF109 transcription factor positively regulates salt tolerance via PalHKT1;2 in Populus alba var. pyramidalis.

Salinity restricts the growth of trees to varying extents, but the regulatory mechanisms involved in their varying salt tolerance are largely unknown. In an effort to elucidate these mechanisms, we identified a total of 99 genes in the Ethylene Responsive Factor (ERF) family of transcription factors and examined their expression patterns under salt stress in Populus alba var. pyramidalis. We found that a B4 group gene, PalERF109, was rapidly induced by salt treatment and preferentially expressed in stems and petioles, where it is probably involved in transport of ions and water in xylem. Overexpression of PalERF109 enhanced the salt tolerance of the poplar, and further analysis showed that it directly upregulated a high-affinity K+transporter (HKT) gene, PalHKT1;2. The results clearly indicate that PalERF109 enhances salt tolerance at least partially through direct activation of PalHKT1;2 and extends understanding of the roles of ERF genes in tree stress responses.