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.

PtoWRKY40 interacts with PtoPHR1-LIKE3 while regulating the phosphate starvation response in poplar.

Plants usually suffer from phosphorus starvation because of the low inorganic phosphate (Pi) status of most soils. To cope with this, plants have evolved an adaptive phosphate starvation response (PSR) which involves both developmental and metabolic changes regulated mainly by PHOSPHATE STARVATION RESPONSE1 (PHR1) and its homologs. Here, we elucidated how perennial woody plants, such as poplars (Populus spp.), respond to low-Pi stress. We first performed RNA-seq analysis of low-Pi-treated poplars and identified PtoWRKY40 is rapidly downregulated and protein degraded after stress. Overexpressing and knocking-down PtoWRKY40 downregulated and upregulated the expression of Pi starvation signaling genes, respectively, such as PHOSPHATE TRANSPORTER1 (PHT1)-type genes and PURPLE ACID PHOSPHATASE genes. PtoWRKY40 bound to the W box in the promoter of several PtoPHT1s and repressed their expression. Moreover, PtoWRKY40 interacted with PtoPHR1-LIKE3 (PtoPHL3), a PHR1 homolog in poplar, to inhibit the latter binding to the P1BS element and thus reduced PtoPHT1s' transcription under Pi-sufficient conditions. However, Pi deficiency decreased PtoWRKY40 abundance and therefore released its inhibition on PHT1s. In conclusion, we have uncovered a PSR mechanism mediated by PtoWRKY40 and PtoPHL3 which regulates Pi content in poplars, deepening our understanding of how poplars adapt to diverse Pi conditions and regulate appropriate responses to maintain Pi homeostasis.

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.

A General Model to Explain Repeated Turnovers of Sex Determination in the Salicaceae.

Dioecy, the presence of separate sexes on distinct individuals, has evolved repeatedly in multiple plant lineages. However, the specific mechanisms by which sex systems evolve and their commonalities among plant species remain poorly understood. With both XY and ZW sex systems, the family Salicaceae provides a system to uncover the evolutionary forces driving sex chromosome turnovers. In this study, we performed a genome-wide association study to characterize sex determination in two Populus species, P. euphratica and P. alba. Our results reveal an XY system of sex determination on chromosome 14 of P. euphratica, and a ZW system on chromosome 19 of P. alba. We further assembled the corresponding sex-determination regions, and found that their sex chromosome turnovers may be driven by the repeated translocations of a Helitron-like transposon. During the translocation, this factor may have captured partial or intact sequences that are orthologous to a type-A cytokinin response regulator gene. Based on results from this and other recently published studies, we hypothesize that this gene may act as a master regulator of sex determination for the entire family. We propose a general model to explain how the XY and ZW sex systems in this family can be determined by the same RR gene. Our study provides new insights into the diversification of incipient sex chromosomes in flowering plants by showing how transposition and rearrangement of a single gene can control sex in both XY and ZW systems.

One AP2/ERF Transcription Factor Positively Regulates Pi Uptake and Drought Tolerance in Poplar.

Drought decreases the inorganic phosphate (Pi) supply of soil, resulting in Pi starvation of plants, but the molecular mechanism of how plants, especially the perennial trees, are tolerant to drought stress and Pi starvation, is still elusive. In this study, we identified an AP2/ERF transcription factor gene, PalERF2, from Populus alba var. pyramidalis, and it was induced by both mannitol treatment and Pi starvation. Overexpressing and knocking-down of PalERF2 both enhanced and attenuated tolerance to drought stress and Pi deficiency compared to WT, respectively. Moreover, the overexpression of PalERF2 up-regulated the expression levels of Pi starvation-induced (PSI) genes and increased Pi uptake under drought conditions; however, its RNAi poplar showed the opposite phenotypes. Subsequent analysis indicated that PalERF2 directly modulated expressions of drought-responsive genes PalRD20 and PalSAG113, as well as PSI genes PalPHL2 and PalPHT1;4, through binding to the DRE motifs on their promoters. These results clearly indicate that poplars can recruit PalERF2 to increase the tolerance to drought and also elevate Pi uptake under drought stress.

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.

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.

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.

Plant Actin-Depolymerizing Factors Possess Opposing Biochemical Properties Arising from Key Amino Acid Changes throughout Evolution.

Functional divergence in paralogs is an important genetic source of evolutionary innovation. Actin-depolymerizing factors (ADFs) are among the most important actin binding proteins and are involved in generating and remodeling actin cytoskeletal architecture via their conserved F-actin severing or depolymerizing activity. In plants, ADFs coevolved with actin, but their biochemical properties are diverse. Unfortunately, the biochemical function of most plant ADFs and the potential mechanisms of their functional divergence remain unclear. Here, in vitro biochemical analyses demonstrated that all 11 ADF genes in Arabidopsis thaliana exhibit opposing biochemical properties. Subclass III ADFs evolved F-actin bundling (B-type) function from conserved F-actin depolymerizing (D-type) function, and subclass I ADFs have enhanced D-type function. By tracking historical mutation sites on ancestral proteins, several fundamental amino acid residues affecting the biochemical functions of these proteins were identified in Arabidopsis and various plants, suggesting that the biochemical divergence of ADFs has been conserved during the evolution of angiosperm plants. Importantly, N-terminal extensions on subclass III ADFs that arose from intron-sliding events are indispensable for the alteration of D-type to B-type function. We conclude that the evolution of these N-terminal extensions and several conserved mutations produced the diverse biochemical functions of plant ADFs from a putative ancestor.

Chromatin accessibility dynamics insight into crosstalk between regulatory landscapes in poplar responses to multiple treatments.

Perennial trees develop and coordinate endogenous response signaling pathways, including their crosstalk and convergence, to cope with various environmental stresses which occur simultaneously in most cases. These processes are involved in gene transcriptional regulations that depend on dynamic interactions between regulatory proteins and corresponding chromatin regions, but the mechanisms remain poorly understood in trees. In this study, we detected chromatin regulatory landscapes of poplar under abscisic acid, methyl jasmonate, salicylic acid and sodium chloride (NaCl) treatment, through integrating ATAC-seq and RNA-seq data. Our results showed that the degree of chromatin accessibility for a given gene is closely related to its expression level. However, unlike the gene expression that shows treatment-specific response patterns, changes in chromatin accessibility exhibit high similarities under these treatments. We further proposed and experimentally validated that a homologous gene copy of RESPONSIVE TO DESICCATION 26 mediates the crosstalk between jasmonic acid and NaCl signaling pathways by directly regulating the stress-responsive genes and that circadian clock-related transcription factors like REVEILLE8 play a central role in response of poplar to these treatments. Overall, our study provides a chromatin insight into the molecular mechanism of transcription regulatory networks in response to different environmental stresses and raises the key roles of the circadian clock of poplar to adapt to adverse environments.

Chromosome-level genome assembly of a xerophytic plant, Haloxylon ammodendron.

Haloxylon ammodendron is a xerophytic perennial shrub or small tree that has a high ecological value in anti-desertification due to its high tolerance to drought and salt stress. Here, we report a high-quality, chromosome-level genome assembly of H. ammodendron by integrating PacBio's high-fidelity sequencing and Hi-C technology. The assembled genome size was 685.4 Mb, of which 99.6% was assigned to nine pseudochromosomes with a contig N50 value of 23.6 Mb. Evolutionary analysis showed that both the recent substantial amplification of long terminal repeat retrotransposons and tandem gene duplication may have contributed to its genome size expansion and arid adaptation. An ample amount of low-GC genes was closely related to functions that may contribute to the desert adaptation of H. ammodendron. Gene family clustering together with gene expression analysis identified differentially expressed genes that may play important roles in the direct response of H. ammodendron to water-deficit stress. We also identified several genes possibly related to the degraded scaly leaves and well-developed root system of H. ammodendron. The reference-level genome assembly presented here will provide a valuable genomic resource for studying the genome evolution of xerophytic plants, as well as for further genetic breeding studies of H. ammodendron.

PtoNF-YC9-SRMT-PtoRD26 module regulates the high saline tolerance of a triploid poplar.

BACKGROUND: Sensing and responding to stresses determine the tolerance of plants to adverse environments. The triploid Chinese white poplar is widely cultivated in North China because of its adaptation to a wide range of habitats including highly saline ones. However, its triploid genome complicates any detailed investigation of the molecular mechanisms underlying its adaptations. RESULTS: We report a haplotype-resolved genome of this triploid poplar and characterize, using reverse genetics and biochemical approaches, a MYB gene, SALT RESPONSIVE MYB TRANSCRIPTION FACTOR (SRMT), which combines NUCLEAR FACTOR Y SUBUNIT C 9 (PtoNF-YC9) and RESPONSIVE TO DESICCATION 26 (PtoRD26), to regulate an ABA-dependent salt-stress response signaling. We reveal that the salt-inducible PtoRD26 is dependent on ABA signaling. We demonstrate that ABA or salt drives PtoNF-YC9 shuttling into the nucleus where it interacts with SRMT, resulting in the rapid expression of PtoRD26 which in turn directly regulates SRMT. This positive feedback loop of SRMT-PtoRD26 can rapidly amplify salt-stress signaling. Interference with either component of this regulatory module reduces the salt tolerance of this triploid poplar. CONCLUSION: Our findings reveal a novel ABA-dependent salt-responsive mechanism, which is mediated by the PtoNF-YC9-SRMT-PtoRD26 module that confers salt tolerance to this triploid poplar. These genes may therefore also serve as potential and important modification targets in breeding programs.

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.

Chromosome-level genome assembly of Sichuan pepper provides insights into apomixis, drought tolerance, and alkaloid biosynthesis.

Sichuan pepper is a commonly used spice in Asian cuisine. Sanshools and wgx-50/gx-50 isolated from it have been shown to possess a wide spectrum of medicinal properties. Here we generated a chromosome-level genome assembly of one Sichuan pepper species Zanthoxylum armatum characterized by drought tolerance and apomixis. Analyses of functionally related genes suggested that increased gene copy number and expression level of drought-tolerant genes might play an important role in improving drought tolerance of Z. armatum. Moreover, a gene encoding an RWP-RK domain-containing protein was shown to contribute to apomixis in Z. armatum, which was further characterized by overexpression in Arabidopsis thaliana. Furthermore, based on gene homology searching and co-expression patterns of metabolite concentration and gene expressions, we identified a number of candidate genes involved in the biosynthesis of sanshools and wgx-50/gx-50. Taken together, our results yield valuable insights for understanding the evolution of apomixis, drought tolerance, and alkaloid biosynthesis in Z. armatum.

Genome-Wide Analysis of the Homeobox Gene Family and Identification of Drought-Responsive Members in Populus trichocarpa.

Homeobox (HB) genes play critical roles in the regulation of plant morphogenesis, growth and development. Here, we identified a total of 156 PtrHB genes from the Populus trichocarpa genome. According to the topologies and taxonomy of the phylogenetic tree constructed by Arabidopsis thaliana HB members, all PtrHB proteins were divided into six subgroups, namely HD-ZIP, ZF-HD, HB-PHD, TALE, WOX and HB-OTHERS. Multiple alignments of conserved homeodomains (HDs) revealed the conserved loci of each subgroup, while gene structure analysis showed similar exon-intron gene structures, and motif analysis indicated the similarity of motif number and pattern in the same subgroup. Promoter analysis indicated that the promoters of PtrHB genes contain a series of cis-acting regulatory elements involved in responding to various abiotic stresses, indicating that PtrHBs had potential functions in these processes. Collinearity analysis revealed that there are 96 pairs of 127 PtrHB genes mainly distributing on Chromosomes 1, 2, and 5. We analyzed the spatio-temporal expression patterns of PtrHB genes, and the virus-induced gene silencing (VIGS) of PtrHB3 gene resulted in the compromised tolerance of poplar seedlings to mannitol treatment. The bioinformatics on PtrHB family and preliminary exploration of drought-responsive genes can provide support for further study of the family in woody plants, especially in drought-related biological processes. It also provides a direction for developing new varieties of poplar with drought resistance. Overall, our results provided significant information for further functional analysis of PtrHB genes in poplar and demonstrated that PtrHB3 is a dominant gene regulating tolerance to water stress treatment in poplar seedlings.

Transcriptional landscape of highly lignified poplar stems at single-cell resolution.

BACKGROUND: Plant secondary growth depends on the activity of the vascular cambium, which produces xylem and phloem. Wood derived from xylem is the most abundant form of biomass globally and has played key socio-economic and subsistence roles throughout human history. However, despite intensive study of vascular development, the full diversity of cell types and the gene networks engaged are still poorly understood. RESULTS: Here, we have applied an optimized protoplast isolation protocol and RNA sequencing to characterize the high-resolution single-cell transcriptional landscape of highly lignified poplar stems. We identify 20 putative cell clusters with a series of novel cluster-specific marker genes and find that these cells are highly heterogeneous based on the transcriptome. Analysis of these marker genes' expression dynamics enables reconstruction of the cell differentiation trajectories involved in phloem and xylem development. We find that different cell clusters exhibit distinct patterns of phytohormone responses and emphasize the use of our data to predict potential gene redundancy and identify candidate genes related to vascular development in trees. CONCLUSIONS: These findings establish the transcriptional landscape of major cell types of poplar stems at single-cell resolution and provide a valuable resource for investigating basic principles of vascular cell specification and differentiation in trees.

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.