Functional Divergence in Orthologous Transcription Factors: Insights from AtCBF2/3/1 and OsDREB1C.

Despite traditional beliefs of orthologous genes maintaining similar functions across species, growing evidence points to their potential for functional divergence. C-repeat binding factors/dehydration-responsive element binding protein 1s (CBFs/DREB1s) are critical in cold acclimation, with their overexpression enhancing stress tolerance but often constraining plant growth. In contrast, a recent study unveiled a distinctive role of rice OsDREB1C in elevating nitrogen use efficiency (NUE), photosynthesis, and grain yield, implying functional divergence within the CBF/DREB1 orthologs across species. Here, we delve into divergent molecular mechanisms of OsDREB1C and AtCBF2/3/1 by exploring their evolutionary trajectories across rice and Arabidopsis genomes, regulatomes, and transcriptomes. Evolutionary scrutiny shows discrete clades for OsDREB1C and AtCBF2/3/1, with the Poaceae-specific DREB1C clade mediated by a transposon event. Genome-wide binding profiles highlight OsDREB1C's preference for GCCGAC compared to AtCBF2/3/1's preference for A/GCCGAC, a distinction determined by R12 in the OsDREB1C AP2/ERF domain. Cross-species multiomic analyses reveal shared gene orthogroups (OGs) and underscore numerous specific OGs uniquely bound and regulated by OsDREB1C, implicated in NUE, photosynthesis, and early flowering, or by AtCBF2/3/1, engaged in hormone and stress responses. This divergence arises from gene gains/losses (∼16.7% to 25.6%) and expression reprogramming (∼62.3% to 66.2%) of OsDREB1C- and AtCBF2/3/1-regulated OGs during the extensive evolution following the rice-Arabidopsis split. Our findings illustrate the regulatory evolution of OsDREB1C and AtCBF2/3/1 at a genomic scale, providing insights on the functional divergence of orthologous transcription factors following gene duplications across species.

Integrative multi-omics analysis of three early diverged rosid species reveals an ancient hierarchical cold-responsive regulatory network.

Elucidating regulators, including transcription factors (TFs) and RNA-binding proteins (RBPs), underlying gene transcriptional and post-transcriptional co-regulatory network is key to understand plant cold responses. Previous studies were mainly conducted on single species, and whether the regulators are conserved across different species remains elusive. Here, we selected three species that diverged at the early evolution of rosids (~99-113 million years ago), performed cold-responsive phylotranscriptome experiments, and integrated chromatin immunoprecipitation- and DNA affinity purification-sequencing (ChIP/DAP-seq) analysis to explore cold-responsive regulators and their regulatory networks. First, we detected over 10,000 cold-induced differentially expressed genes (DEGs) and alternative splicing genes (DASGs) in each species. Among the DEGs, a set of TFs and RBPs were conserved in rosid cold response. Compared to TFs, RBPs displayed a delayed cold-responsive pattern, implying a hierarchical regulation of DEGs and DASGs. By integrating DEGs and DASGs, we identified 259 overlapping DE-DASG orthogroups (closely-related homologs) that were cold-regulated at both transcriptional and post-transcriptional levels in all three studied species. Notably, pathway analysis on each of the DEGs, DASGs, and DE-DASGs in the three species showed a common enrichment connected to the circadian rhythm. Evidently, 226 cold-responsive genes were directly targeted by at least two circadian rhythm components (CCA1, LHY, RV4, RVE7, and RVE8). Finally, we revealed an ancient hierarchy of cold-responsive regulatory networks at transcriptional and post-transcriptional levels launched by circadian components in rosids. Altogether, this study sheds light on conserved regulators underlying cold-responsive regulatory networks across rosid species, despite a long evolutionary history after their divergence.

Innovations and stepwise evolution of CBFs/DREB1s and their regulatory networks in angiosperms.

The C-repeat binding factors/dehydration-responsive element binding protein 1s (CBFs/DREB1s) have been identified as major regulators of cold acclimation in many angiosperm plants. However, their origin and evolutionary process associated to cold responsiveness are still lacking. By integrating multi-omics data of genomes, transcriptomes, and CBFs/DREB1s genome-wide binding profiles, we unveil the origin and evolution of CBFs/DREB1s and their regulatory network. Gene collinearity and phylogeny analyses show that CBF/DREB1 is an innovation evolved from tandem duplication-derived DREB III gene. A subsequent event of ε-whole genome duplication led to two CBF/DREB1 archetypes (Clades I and II) in ancient angiosperms. In contrast to cold-insensitivity of Clade I and their parent DREB III genes, Clade II evolved a further innovation in cold-sensitive response and was stepwise expanded in eudicots and monocots by independent duplications. In geological time, the duplication events were mainly enriched around the Cretaceous-Paleogene (K-Pg) boundary and/or in the Late Cenozoic Ice Age, when the global average temperature significantly decreased. Consequently, the duplicated CBF/DREB1 genes contributed to the rewiring of CBFs/DREB1s-regulatory network for cold tolerance. Altogether, our results highlight an origin and convergent evolution of CBFs/DREB1s and their regulatory network probably for angiosperms adaptation to global cooling.

Orthogroup and phylotranscriptomic analyses identify transcription factors involved in the plant cold response: A case study of Arabidopsis BBX29.

C-repeat binding factors (CBFs) are well-known transcription factors (TFs) that regulate plant cold acclimation. RNA sequencing (RNA-seq) data from diverse plant species provide opportunities to identify other TFs involved in the cold response. However, this task is challenging because gene gain and loss has led to an intertwined community of co-orthologs and in-paralogs between and within species. Using orthogroup (closely related homologs) analysis, we identified 10,549 orthogroups in five representative eudicots. A phylotranscriptomic analysis of cold-treated seedlings from eudicots identified 35 high-confidence conserved cold-responsive transcription factor orthogroups (CoCoFos). These 35 CoCoFos included the well-known cold-responsive regulators CBFs, HSFC1, ZAT6/10, and CZF1 among others. We used Arabidopsis BBX29 for experimental validation. Expression and genetic analyses showed that cold-induction of BBX29 is CBF- and abscisic acid-independent, and BBX29 is a negative regulator of cold tolerance. Integrative RNA-seq and Cleavage Under Targets and Tagmentation followed by sequencing analyses revealed that BBX29 represses a set of cold-induced TFs (ZAT12, PRR9, RVE1, MYB96, etc.). Altogether, our analysis yielded a library of eudicot CoCoFos and demonstrated that BBX29 is a negative regulator of cold tolerance in Arabidopsis.

Preparation and Characterization of the Forward Osmosis Membrane Modified by MXene Nano-Sheets.

The Forward Osmosis (FO) membrane was the core of FO technology. Obtaining a high water flux while maintaining a low reverse solute flux has historically been considered the gold standard for a perfect FO membrane. In a thin-film composite FO membrane, the performance of the membrane was determined not only by the material and structure of the porous support layer but also by the structural and chemical properties of the active selective layer. Researchers have selected numerous sorts of materials for the FO membranes in recent years and have produced exceptional achievements. Herein, the performance of the modified FO membrane constructed by introducing new two-dimensional nanomaterial MXene nano-sheets to the interfacial polymerization process was investigated, and the performance of these modified membranes was investigated using a variety of characterization and testing methods. The results revealed that the MXene nano-sheets played an important role in improving the performance of the FO membrane. Because of the hydrophilic features of the MXene nano-sheets, the membrane structure may be tuned within a specific concentration range, and the performance of the modified FO membrane has been significantly enhanced accordingly. The optimal membrane water flux was boosted by around 80%, while its reverse solute flux was kept to a minimum of the resultant membranes. It showed that the addition of MXene nanosheets to the active selective layer could improve the performance of the FO membrane, and this method showed promising application prospects.

Preparation and Characterization of a Thin-Film Composite Membrane Modified by MXene Nano-Sheets.

MXene nano-sheets were introduced into a thin-film composite membrane (TFC) to reduce the mass transfer resistance (concentration polarization) and improve the membrane performance. The process entailed dissolving the MXene nano-sheets in a membrane casting solution using the blending method and introducing them into the porous support layer to prepare a modified thin-film composite forward osmosis (TFC-FO) membrane. The results showed that the water contact angle decreased by about 16%, indicating that the hydrophilicity was strengthened, and the O/N ratio of the active selective layer decreased by 13%, indicating an increased degree of crosslinking, thereby demonstrating that the introduction of MXene nano-sheets changed the properties of the membrane and played a positive role in its physicochemical properties. In contrast to the unmodified TFC-FO membrane, the modified membrane had a slightly higher reverse solute flux, while its water flux increased by about 80%. Its specific reverse osmosis flux was also significantly optimized (only 0.63 g/L). In conclusion, adding MXene nanosheets to TFC-FO membranes led to the modified membranes with better mass transfer, lessened internal concentration polarization (ICP), and better osmotic separation.

Convergent evolution of AP2/ERF III and IX subfamilies through recurrent polyploidization and tandem duplication during eudicot adaptation to paleoenvironmental changes.

Whole-genome duplication (WGD or polyploidization) has been suggested as a genetic contributor to angiosperm adaptation to environmental changes. However, many eudicot lineages did not undergo recent WGD (R-WGD) around and/or after the Cretaceous-Paleogene (K-Pg) boundary, times of severe environmental changes; how those plants survived has been largely ignored. Here, we collected 22 plants from major branches of the eudicot phylogeny and classified them into two groups according to the occurrence or absence of R-WGD: 12 R-WGD-containing plants (R-WGD-Y) and 10 R-WGD-lacking plants (R-WGD-N). Subsequently, we identified 496 gene-rich families in R-WGD-Y and revealed that members of the AP2/ERF transcription factor family were convergently over-retained after R-WGDs and showed exceptional cold stimulation. The evolutionary trajectories of the AP2/ERF family were then compared between R-WGD-Y and R-WGD-N to reveal convergent expansions of the AP2/ERF III and IX subfamilies through recurrent independent WGDs and tandem duplications (TDs) after the radiation of the plants. The expansions showed coincident enrichments in- times around and/or after the K-Pg boundary, when global cooling was a major environmental stressor. Consequently, convergent expansions and co-retentions of AP2/ERF III C-repeat binding factor (CBF) duplicates and their regulons in different eudicot lineages contributed to the rewiring of cold-specific regulatory networks. Moreover, promoter analysis of cold-responsive AP2/ERF genes revealed an underlying cis-regulatory code (G-box: CACGTG). We propose a seesaw model of WGDs and TDs in the convergent expansion of AP2/ERF III and IX genes that has contributed to eudicot adaptation during paleoenvironmental changes, and we suggest that TD may be a reciprocal/alternative mechanism for genetic innovation in plants that lack WGD.

Innovation and Emerging Roles of Populus trichocarpa TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR Transcription Factors in Abiotic Stresses by Whole-Genome Duplication.

The TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) family proteins are plant-specific transcription factors that have been well-acknowledged for designing the architectures of plant branch, shoot, and inflorescence. However, evidence for their innovation and emerging role in abiotic stress has been lacking. In this study, we identified a total of 36 TCP genes in Populus trichocarpa, 50% more than that in Arabidopsis (i.e., 24). Comparative intra-genomes showed that such significant innovation was mainly due to the most recent whole genome duplication (rWGD) in Populus lineage around Cretaceous-Paleogene (K-Pg) boundary after the divergence from Arabidopsis. Transcriptome analysis showed that the expressions of PtrTCP genes varied among leaf, stem, and root, and they could also be elaborately regulated by abiotic stresses (e.g., cold and salt). Moreover, co-expression network identified a cold-associated regulatory module including PtrTCP31, PtrTCP10, and PtrTCP36. Of them, PtrTCP10 was rWGD-duplicated from PtrTCP31 and evolved a strong capability of cold induction, which might suggest a neofunctionalization of PtrTCP genes and contribute to the adaptation of Populus lineage during the Cenozoic global cooling. Evidentially, overexpression of PtrTCP10 into Arabidopsis increased freezing tolerance and salt susceptibility. Integrating co-expression network and cis-regulatory element analysis confirmed that PtrTCP10 can regulate the well-known cold- and salt-relevant genes (e.g., ZAT10, GolS2, and SOS1), proving that PtrTCP10 is an evolutionary innovation in P. trichocarpa response to environmental changes. Altogether, our results provide evidence of the rWGD in P. trichocarpa responsible for the innovation of PtrTCP genes and their emerging roles in environmental stresses.

Inverse design of photonic meta-structure for beam collimation in on-chip sensing.

Designed or patterned structured surfaces, metasurfaces, enable the miniaturization of complex arrangements of optical elements on a plane. Most of the existing literature focuses on miniaturizing the optical detection; little attention is directed to on-chip optical excitation. In this work, we design a metasurface to create a planar integrated photonic source beam collimator for use in on-chip optofluidic sensing applications. We use an iterative inverse design approach in order to optimize the metasurface to achieve a target performance using gradient descent method. We then fabricate beam collimators and experimentally compare performance characteristics with conventional uniform binary grating-based photonic beam diffractors. The optimal design enhances the illumination power by a factor of 5. The reinforced beam is more uniform with 3 dB beam spot increased almost ~ 3 times for the same device footprint area. The design approach will be useful in on-chip applications of fluorescence imaging, Raman, and IR spectroscopy and will enable better multiplexing of light sources for high throughput biosensing.