RAV1 (Related to ABI3/VP1) is a plant‐specific B3 and AP2 domain‐containing transcription factor that acts as a negative regulator of growth in many plant species. The expression of RAV1 is downregulated by brassinosteroids (BRs); large‐scale transcriptome analyses have shown that the expression of RAV1 was previously targeted by BRI1‐EMS‐SUPPRESOR1 (BES1) and BRASSINAZOLE‐RESISTANT1 (BZR1), which are critical transcription factors for the BR‐ signaling process. Using RAV1‐overexpressing transgenic plants, we showed that RAV1 overexpression reduced the BR signaling capacity, resulting in the downregulation of BR biosynthetic genes and BES1 expression. Furthermore, we demonstrated that BES1, not BZR1, is directly bound to the RAV1 promoter and repressed RAV1 expression, and vice versa; RAV1 is also bound to the BES1 promoter and repressed BES1 expression. This mutual inhibition was specific to RAV1 and BES1 because RAV1 exhibited binding activity to the BZR1 promoter but did not repress BZR1 expression. We observed that constitutively activated BR signaling phenotypes in bes1‐D were attenuated by the repression of endogenous BES1 expression in transgenic bes1‐D plants overexpressing RAV1. RNA‐ sequencing analysis of RAV1‐overexpressing transgenic plants and bes1‐D mutant plants revealed differentially expressed genes by RAV1 and BES1 and genes that were oppositely co‐regulated by RAV1 and BES1. RAV1 and BES1 regulated different transcriptomes but co‐regulated a specific set of genes responsible for the balance between growth and defense. These results suggested that the mutual inhibitory transcriptional activities of RAV1 and BES1 provide fine regulatory mechanisms for plant growth and development.

For adaptation to ever‐changing environments, plants have evolved elaborate metabolic systems coupled to a regulatory network for optimal growth and defense. Regulation of plant secondary metabolic pathways such as glucosinolates (GSLs) by defense phytohormones in response to different stresses and nutrient deficiency has been intensively investigated, while how growth‐promoting hormone balances plant secondary and primary metabolism has been largely unexplored. Here, we found that growth‐promoting hormone brassinosteroid (BR) inhibits GSLs accumulation while enhancing biosynthesis of primary sulfur metabolites, including cysteine (Cys) and glutathione (GSH) both in Arabidopsis and Brassica crops, fine‐tuning secondary and primary sulfur metabolism to promote plant growth. Furthermore, we demonstrate that of BRASSINAZOLE RESISTANT 1 (BZR1), the central component of BR signaling, exerts distinct transcriptional inhibition regulation on indolic and aliphatic GSL via direct MYB51 dependent repression of indolic GSL biosynthesis, while exerting partial MYB29 dependent repression of aliphatic GSL biosynthesis. Additionally, BZR1 directly activates the transcription of APR1 and APR2 which encodes rate‐limiting enzyme adenosine 5′‐phosphosulfate reductases in the primary sulfur metabolic pathway. In summary, our findings indicate that BR inhibits the biosynthesis of GSLs to prioritize sulfur usage for primary metabolites under normal growth conditions. These findings expand our understanding of BR promoting plant growth from a metabolism perspective.

The Gametophyte factor1 (Ga1) locus in maize confers unilateral cross‐incompatibility (UCI), and it is controlled by both pollen and silk‐specific determinants. Although the Ga1 locus has been reported for more than a century and is widely utilized in maize breeding programs, only the pollen‐specific ZmGa1P has been shown to function as a male determinant; thus, the genomic structure of the Ga1 locus and all the determinants that control UCI at this locus have not yet been fully characterized. Here, we used map‐based cloning to confirm the determinants of UCI at the Ga1 locus and maize pan‐genome sequence data to characterize the genomic structure of the Ga1 locus. The Ga1 locus comprises one silk‐expressed pectin methylesterase gene (PME, ZmGa1F) and eight pollen‐ expressed PMEs (ZmGa1P and ZmGa1PL1‐7). Knockout of ZmGa1F in Ga1/Ga1 lines leads to the complete loss of the female barrier function. The expression of individual ZmGa1PL genes in a ga1/ga1 background endows ga1 pollen with the ability to overcome the female barrier of the Ga1 locus. These findings, combined with genomic data and genetic analyses, indicate that the Ga1 locus is modulated by a single female determinant and multiple male determinants, which are tightly linked. The results of this study provide valuable insights into the genomic structure of the Ga2 and Tcb1 loci and will aid applications of these loci in maize breeding programs.

Peptidyl‐prolyl isomerase‐like 1 (PPIL1) is associated with the human spliceosome complex. However, its function in pre‐mRNA splicing remains unclear. In this study, we show that Arabidopsis thaliana CYCLOPHILIN 18‐2 (AtCYP18‐2), a PPIL1 homolog, plays an essential role in heat tolerance by regulating pre‐mRNA splicing. Under heat stress conditions, AtCYP18‐2 expression was upregulated in mature plants and GFP‐tagged AtCYP18‐2 redistributed to nuclear and cytoplasmic puncta. We determined that AtCYP18‐2 interacts with several spliceosome complex B^ACT components in nuclear puncta and is primarily associated with the small nuclear RNAs U5 and U6 in response to heat stress. The AtCYP18‐2 loss‐of‐function allele cyp18‐2 engineered by CRISPR/Cas9‐mediated gene editing exhibited a hypersensitive phenotype to heat stress relative to the wild type. Moreover, global transcriptome profiling showed that the cyp18‐2 mutation affects alternative splicing of heat stress–responsive genes under heat stress conditions, particularly intron retention (IR). The abundance of most intron‐containing transcripts of a subset of genes essential for thermotolerance decreased in cyp18‐2 compared to the wild type. Furthermore, the intron‐containing transcripts of two heat stress‐related genes, HEAT SHOCK PROTEIN 101 (HSP101) and HEAT SHOCK FACTOR A2 (HSFA2), produced functional proteins. HSP101‐IR‐GFP localization was responsive to heat stress, and HSFA2‐III‐IR interacted with HSF1 and HSP90.1 in plant cells. Our findings reveal that CYP18‐2 functions as a splicing factor within the B^ACT spliceosome complex and is crucial for ensuring the production of adequate levels of alternatively spliced transcripts to enhance thermotolerance.

Orchidaceae (with >28,000 orchid species) are one of the two largest plant families, with economically and ecologically important species, and occupy global and diverse niches with primary distribution in rainforests. Among orchids, 70% grow on other plants as epiphytes; epiphytes contribute up to ~50% of the plant diversity in rainforests and provide food and shelter for diverse animals and microbes, thereby contributing to the health of these ecosystems. Orchids account for over two-thirds of vascular epiphytes and provide an excellent model for studying evolution of epiphytism. Extensive phylogenetic studies of Orchidaceae and subgroups have ;been crucial for understanding relationships among many orchid lineages, although some uncertainties remain. For example, in the largest subfamily Epidendroideae with nearly all epiphytic orchids, relationships among some tribes and many subtribes are still controversial, hampering evolutionary analyses of epiphytism. Here we obtained 1,450 low-copy nuclear genes from 610 orchid species, including 431 with newly generated transcriptomes, and used them for the reconstruction of robust Orchidaceae phylogenetic trees with highly supported placements of tribes and subtribes. We also provide generally well-supported phylogenetic placements of 131 genera and 437 species that were not sampled by previous plastid and nuclear phylogenomic studies. Molecular clock analyses estimated the Orchidaceae origin at ~132 million years ago (Ma) and divergences of most subtribes from 52 to 29?Ma. Character reconstruction supports at least 14 parallel origins of epiphytism; one such origin was placed at the most recent common ancestor of ~95% of epiphytic orchids and linked to modern rainforests. Ten occurrences of rapid increase in the diversification rate were detected within Epidendroideae near and after the K-Pg boundary, contributing to ~80% of the Orchidaceae diversity. This study provides a robust and the largest family-wide Orchidaceae nuclear phylogenetic tree thus far and new insights into the evolution of epiphytism in vascular plants.

The phytohormone salicylic acid (SA) regulates biotic and abiotic stress responses in plants. Two distinct biosynthetic pathways for SA have been well documented in plants: the isochorismate (IC) pathway in the chloroplast and the phenylalanine ammonia‐lyase (PAL) pathway in the cytosol. However, there has been no solid evidence that the PAL pathway contributes to SA biosynthesis. Here, we report that feeding Arabidopsis thaliana with Ring‐¹³C‐labeled phenylalanine (¹³C₆‐Phe) resulted in incorporation of the ¹³C label not into SA, but into its isomer 4‐hydroxybenzoic acid (4‐HBA) instead. We obtained similar results when feeding ¹³C₆‐Phe to the SA‐deficient ics1 ics2 mutant and the SA‐hyperaccumulating mutant s3h s5h. Notably, we detected ¹³C₆‐SA when ¹³C₆‐benzoic acid (BA) was provided, suggesting that SA can be synthesized from BA. Furthermore, despite the substantial accumulation of SA upon pathogen infection, we did not observe incorporation of ¹³C label from Phe into SA. We also did not detect ¹³C₆‐SA in PAL‐overexpressing lines in the kfb01 kfb02 kfb39 kfb50 background after being fed ¹³C₆‐ Phe, although endogenous PAL levels were dramatically increased. Based on these combined results, we propose that SA biosynthesis is not from Phe in Arabidopsis. These results have important implications for our understanding of the SA biosynthetic pathway in land plants.

In Arabidopsis, although studies have demonstrated that phytochrome A (phyA) and phyB are involved in blue light signaling, how blue light‐ activated phytochromes modulate the activity of the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)‐SUPPRESSOR OF PHYA‐105 (SPA1) E3 complex remains largely unknown. Here, we show that phyA responds to early and weak blue light, whereas phyB responds to sustainable and strong blue light. Activation of both phyA and phyB by blue light inhibits SPA1 activity. Specifically, blue light irradiation promoted the nuclear import of both phytochromes to stimulate their binding to SPA1, abolishing SPA1's interaction with LONG HYPOCOTYL 5 (HY5) to release HY5, which promoted seedling photomorphogenesis.

Halophytes complete their life cycles in saline environments. The recretohalophyte Limonium bicolor has evolved a specialized salt secretory structure, the salt gland, which excretes Na⁺ to avoid salt damage. Typical L. bicolor salt glands consist of 16 cells with four fluorescent foci and four secretory pores. Here, we describe a special type of salt gland at the base of the L. bicolor leaf petiole named bracelet salt glands due to their beaded‐bracelet‐ like shape of blue auto‐fluorescence. Bracelet salt glands contain more than 16 cells and more than four secretory pores. Leaf disc secretion measurements and non‐invasive micro‐test techniques indicated that bracelet salt glands secrete more salt than normal salt glands, which helps maintain low Na⁺ levels at the leaf blade to protect the leaf. Cytokinin treatment induced bracelet salt gland differentiation, and the developed ones showed no further differentiation when traced with a living fluorescence microscopy imager, even though new salt gland development and leaf expansion were observed. Transcriptome revealed a NAC transcription factor gene that participates in bracelet salt gland development, as confirmed by its genome editing and overexpression in L. bicolor. These findings shed light on bracelet salt gland development and may facilitate the engineering of salt‐tolerant crops.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐related nuclease 9 (Cas9) system enables precise, simple editing of genes in many animals and plants. However, this system has not been applied to rose (Rosa hybrida) due to the genomic complexity and lack of an efficient transformation technology for this plant. Here, we established a platform for screening single‐guide RNAs (sgRNAs) with high editing efficiency for CRISPR/Cas9‐mediated gene editing in rose using suspension cells. We used the Arabidopsis thaliana U6‐29 promoter, which showed high activity for driving sgRNA expression, to modify the CRISPR/Cas9 system. We used our highly efficient optimized CRISPR/Cas9 system to successfully edit RhEIN2, encoding an indispensable component of the ethylene signaling pathway, resulting in ethylene insensitivity in rose. Our optimized CRISPR/Cas9 system provides a powerful toolbox for functional genomics, molecular breeding, and synthetic biology in rose.

Cotton (Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However, genotype dependence is one of the key bottlenecks in generating transgenic and gene‐edited cotton plants through either particle bombardment or Agrobacterium‐mediated transformation. Here, we developed a shoot apical meristem (SAM) cell‐ mediated transformation system (SAMT) that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton (Gossypium hirsutum), Sea island cotton (Gossypium barbadense), and Asiatic cotton (Gossypium arboreum). Through SAMT, we successfully introduced two foreign genes, GFP and RUBY, into SAM cells of some recalcitrant cotton genotypes. Within 2–3 months, transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants. The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP‐ and RUBY‐positive plants, as well as in their progenies, indicating that the transgenes were stably integrated into the genome and transmitted to the next generation. Furthermore, using SAMT, we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9‐mediated genome editing. In summary, the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene‐edited plants for genetic improvement of recalcitrant cotton varieties.

Arms race co-evolution of plant–pathogen interactions evolved sophisticated recognition mechanisms between host immune receptors and pathogen effectors. Different allelic haplotypes of an immune receptor in the host mount distinct recognition against sequence or non-sequence related effectors in pathogens. We report the molecular characterization of the Piks allele of the rice immune receptor Pik against rice blast pathogen, which requires two head-to-head arrayed nucleotide-binding sites and leucine-rich repeat proteins. Like other Pik alleles, both Piks-1 and Piks-2 are necessary and sufficient for mediating resistance. However, unlike other Pik alleles, Piks does not recognize any known AvrPik variants of Magnaporthe oryzae. Sequence analysis of the genome of an avirulent isolate V86010 further revealed that its cognate avirulence (Avr) gene most likely has no significant sequence similarity to known AvrPik variants. Piks-1 and Pikm-1 have only two amino acid differences within the integrated heavy metal-associated (HMA) domain. Pikm-HMA interacts with AvrPik-A, -D, and -E in vitro and in vivo, whereas Piks-HMA does not bind any AvrPik variants. Characterization of two amino acid residues differing Piks-1 from Pikm-1 reveal that Piks-E229Q derived from the exchange of Glu229 to Gln229 in Piks-1 gains recognition specificity against AvrPik-D but not AvrPik-A or -E, indicating that Piks-E229Q partially restores the Pikm spectrum. By contrast, Piks-A261V derived from the exchange of Ala261 to Val261 in Piks-1 retains Piks recognition specificity. We conclude that Glu229 in Piks-1 is critical for Piks breaking the canonical Pik/AvrPik recognition pattern. Intriguingly, binding activity and ectopic cell death induction is maintained between Piks-A261V and AvrPik-D, implying that positive outcomes from ectopic assays might be insufficient to deduce its immune activity against the relevant effectors in rice and rice blast interaction.

Moderate stimuli in mitochondria improve wide-ranging stress adaptability in animals, but whether mitochondria play similar roles in plants is largely unknown. Here, we report the enhanced stress adaptability of S-type cytoplasmic male sterility (CMS-S) maize and its association with mild expression of sterilizing gene ORF355. A CMS-S maize line exhibited superior growth potential and higher yield than those of the near-isogenic N-type line in saline fields. Moderate expression of ORF355 induced the accumulation of reactive oxygen species and activated the cellular antioxidative defense system. This adaptive response was mediated by elevation of the nicotinamide adenine dinucleotide concentration and associated metabolic homeostasis. Metabolome analysis revealed broad metabolic changes in CMS-S lines, even in the absence of salinity stress. Metabolic products associated with amino acid metabolism and galactose metabolism were substantially changed, which underpinned the alteration of the antioxidative defense system in CMS-S plants. The results reveal the ORF355-mediated superior stress adaptability in CMS-S maize and might provide an important route to developing salt-tolerant maize varieties.

Whole-genome genotyping methods are important for breeding. However, it has been challenging to develop a robust method for simultaneous foreground and background genotyping that can easily be adapted to different genes and species. In our study, we accidently discovered that in adapter ligation-mediated PCR, the amplification by primer-template mismatched annealing (PTMA) along the genome could generate thousands of stable PCR products. Based on this observation, we consequently developed a novel method for simultaneous foreground and background integrated genotyping by sequencing (FBI-seq) using one specific primer, in which foreground genotyping is performed by primer-template perfect annealing (PTPA), while background genotyping employs PTMA. Unlike DNA arrays, multiple PCR, or genome target enrichments, FBI-seq requires little preliminary work for primer design and synthesis, and it is easily adaptable to different foreground genes and species. FBI-seq therefore provides a prolific, robust, and accurate method for simultaneous foreground and background genotyping to facilitate breeding in the post-genomics era.

In contrast to large-effect qualitative disease resistance, quantitative disease resistance (QDR) exhibits partial and generally durable resistance and has been extensively utilized in crop breeding. The molecular mechanisms underlying QDR remain largely unknown but considerable progress has been made in this area in recent years. In this review, we summarize the genes that have been associated with plant QDR and their biological functions. Many QDR genes belong to the canonical resistance gene categories with predicted functions in pathogen perception, signal transduction, phytohormone homeostasis, metabolite transport and biosynthesis, and epigenetic regulation. However, other “atypical” QDR genes are predicted to be involved in processes that are not commonly associated with disease resistance, such as vesicle trafficking, molecular chaperones, and others. This diversity of function for QDR genes contrasts with qualitative resistance, which is often based on the actions of nucleotide-binding leucine-rich repeat (NLR) resistance proteins. An understanding of the diversity of QDR mechanisms and of which mechanisms are effective against which classes of pathogens will enable the more effective deployment of QDR to produce more durably resistant, resilient crops.

Soybean (Glycine max) is a major source of plant protein and oil. Soybean breeding has benefited from advances in functional genomics. In particular, the release of soybean reference genomes has advanced our understanding of soybean adaptation to soil nutrient deficiencies, the molecular mechanism of symbiotic nitrogen (N) fixation, biotic and abiotic stress tolerance, and the roles of flowering time in regional adaptation, plant architecture, and seed yield and quality. Nevertheless, many challenges remain for soybean functional genomics and molecular breeding, mainly related to improving grain yield through high-density planting, maize–soybean intercropping, taking advantage of wild resources, utilization of heterosis, genomic prediction and selection breeding, and precise breeding through genome editing. This review summarizes the current progress in soybean functional genomics and directs future challenges for molecular breeding of soybean.

Asymmetric cell division (ACD) is a fundamental process that generates new cell types during development in eukaryotic species. In plant development, post-embryonic organogenesis driven by ACD is universal and more important than in animals, in which organ pattern is preset during embryogenesis. Thus, plant development provides a powerful system to study molecular mechanisms underlying ACD. During the past decade, tremendous progress has been made in our understanding of the key components and mechanisms involved in this important process in plants. Here, we present an overview of how ACD is determined and regulated in multiple biological processes in plant development and compare their conservation and specificity among different model cell systems. We also summarize the molecular roles and mechanisms of the phytohormones in the regulation of plant ACD. Finally, we conclude with the overarching paradigms and principles that govern plant ACD and consider how new technologies can be exploited to fill the knowledge gaps and make new advances in the field.

Brassinosteroids (BRs) and abscisic acid (ABA) are essential regulators of plant growth and stress tolerance. Although the antagonistic interaction of BRs and ABA is proposed to ensure the balance between growth and defense in model plants, the crosstalk between BRs and ABA in response to chilling in tomato (Solanum lycopersicum), a warm-climate horticultural crop, is unclear. Here, we determined that overexpression of the BR biosynthesis gene DWARF (DWF) or the key BR signaling gene BRASSINAZOLE-RESISTANT1 (BZR1) increases ABA levels in response to chilling stress via positively regulating the expression of the ABA biosynthesis gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE1 (NCED1). BR-induced chilling tolerance was mostly dependent on ABA biosynthesis. Chilling stress or high BR levels decreased the abundance of BRASSINOSTEROID-INSENSITIVE2 (BIN2), a negative regulator of BR signaling. Moreover, we observed that chilling stress increases BR levels and results in the accumulation of BZR1. BIN2 negatively regulated both the accumulation of BZR1 protein and chilling tolerance by suppressing ABA biosynthesis. Our results demonstrate that BR signaling positively regulates chilling tolerance via ABA biosynthesis in tomato. The study has implications in production of warm-climate crops in horticulture.

The ethylene insensitive 3/ethylene insensitive 3-like (EIN3/EIL) plays an indispensable role in fruit ripening. However, the regulatory mechanism that links post-translational modification of EIN3/EIL to fruit ripening is largely unknown. Here, we studied the expression of 13 MaEIL genes during banana fruit ripening, among which MaEIL9 displayed higher enhancement particularly in the ripening stage. Consistent with its transcript pattern, abundance of MaEIL9 protein gradually increased during the ripening process, with maximal enhancement in the ripening. DNA affinity purification (DAP)-seq analysis revealed that MaEIL9 directly targets a subset of genes related to fruit ripening, such as the starch hydrolytic genes MaAMY3D and MaBAM1. Stably overexpressing MaEIL9 in tomato fruit hastened fruit ripening, whereas transiently silencing this gene in banana fruit retarded the ripening process, supporting a positive role of MaEIL9 in fruit ripening. Moreover, oxidation of methionines (Met-129, Met-130, and Met-282) in MaEIL9 resulted in the loss of its DNA-binding capacity and transcriptional activation activity. Importantly, we identified MaEIL9 as a potential substrate protein of methionine sulfoxide reductase A MaMsrA4, and oxidation of Met-129, Met-130, and Met-282 in MaEIL9 could be restored by MaMsrA4. Collectively, our findings reveal a novel regulatory network controlling banana fruit ripening, which involves MaMsrA4-mediated redox regulation of the ethylene signaling component MaEIL9.

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in eukaryotic cells that directly regulates abundance of plasma membrane proteins. Clathrin triskelia are composed of clathrin heavy chains (CHCs) and light chains (CLCs), and the phytohormone auxin differentially regulates membrane-associated CLCs and CHCs, modulating the endocytosis and therefore the distribution of auxin efflux transporter PIN-FORMED2 (PIN2). However, the molecular mechanisms by which auxin regulates clathrin are still poorly understood. Transmembrane kinase (TMKs) family proteins are considered to contribute to auxin signaling and plant development; it remains unclear whether they are involved in PIN transport by CME. We assessed TMKs involvement in the regulation of clathrin by auxin, using genetic, pharmacological, and cytological approaches including live-cell imaging and immunofluorescence. In tmk1 mutant seedlings, auxin failed to rapidly regulate abundance of both CHC and CLC and to inhibit PIN2 endocytosis, leading to an impaired asymmetric distribution of PIN2 and therefore auxin. Furthermore, TMK3 and TMK4 were shown not to be involved in regulation of clathrin by auxin. In summary, TMK1 is essential for auxin-regulated clathrin recruitment and CME. TMK1 therefore plays a critical role in the establishment of an asymmetric distribution of PIN2 and an auxin gradient during root gravitropism.

Integration of light signaling and diverse abiotic stress responses contribute to plant survival in a changing environment. Some reports have indicated that light signals contribute a plant's ability to deal with heat, cold, and stress. However, the molecular link between light signaling and the salt-response pathways remains unclear. We demonstrate here that increasing light intensity elevates the salt stress tolerance of plants. Depletion of HY5, a key component of light signaling, causes Arabidopsis thaliana to become salinity sensitive. Interestingly, the small heat shock protein (sHsp) family genes are upregulated in hy5-215 mutant plants, and HsfA2 is commonly involved in the regulation of these sHsps. We found that HY5 directly binds to the G-box motifs in the HsfA2 promoter, with the cooperation of HISTONE DEACETYLASE 9 (HDA9), to repress its expression. Furthermore, the accumulation of HDA9 and the interaction between HY5 and HDA9 are significantly enhanced by salt stress. On the contrary, high temperature triggers HY5 and HDA9 degradation, which leads to dissociation of HY5-HDA9 from the HsfA2 promoter, thereby reducing salt tolerance. Under salt and heat stress conditions, fine tuning of protein accumulation and an interaction between HY5 and HDA9 regulate HsfA2 expression. This implies that HY5, HDA9, and HsfA2 play important roles in the integration of light signaling with salt stress and heat shock response.

Advances in plant phenotyping technologies are dramatically reducing the marginal costs of collecting multiple phenotypic measurements across several time points. Yet, most current approaches and best statistical practices implemented to link genetic and phenotypic variation in plants have been developed in an era of single-time-point data. Here, we used time-series phenotypic data collected with an unmanned aircraft system for a large panel of soybean (Glycine max (L.) Merr.) varieties to identify previously uncharacterized loci. Specifically, we focused on the dissection of canopy coverage (CC) variation from this rich data set. We also inferred the speed of canopy closure, an additional dimension of CC, from the time-series data, as it may represent an important trait for weed control. Genome-wide association studies (GWASs) identified 35 loci exhibiting dynamic associations with CC across developmental stages. The time-series data enabled the identification of 10 known flowering time and plant height quantitative trait loci (QTLs) detected in previous studies of adult plants and the identification of novel QTLs influencing CC. These novel QTLs were disproportionately likely to act earlier in development, which may explain why they were missed in previous single-time-point studies. Moreover, this time-series data set contributed to the high accuracy of the GWASs, which we evaluated by permutation tests, as evidenced by the repeated identification of loci across multiple time points. Two novel loci showed evidence of adaptive selection during domestication, with different genotypes/haplotypes favored in different geographic regions. In summary, the time-series data, with soybean CC as an example, improved the accuracy and statistical power to dissect the genetic basis of traits and offered a promising opportunity for crop breeding with quantitative growth curves.

The salinization of irrigated land affects agricultural productivity. HIGH-AFFINITY POTASSIUM (K⁺) TRANSPORTER 1;5 (OsHKT1;5)-dependent sodium (Na⁺) transport is a key salt tolerance mechanism during rice growth and development. Using a previously generated high-throughput activation tagging-based T-DNA insertion mutant pool, we isolated a mutant exhibiting salt stress-sensitive phenotype, caused by a reduction in OsHKT1;5 transcripts. The salt stress-sensitive phenotype of this mutant results from the loss of function of OsDNAJ15, which encodes plasma membrane-localized heat shock protein 40 (Hsp40). osdnaj15 loss-of-function mutants show decreased plant height, increased leaf angle, and reduced grain number caused by shorter panicle length and fewer branches. On the other h'and, OsDNAJ15-overexpression plants showed salt stress-tolerant phenotypes. Intriguingly, salt stress facilitates the nuclear relocation of OsDNAJ15 so that it can interact with OsBAG4, and OsDNAJ15 and OsBAG4 synergistically facilitate the DNA-binding activity of OsMYB106 to positively regulate the expression of OsHKT1;5. Overall, our results reveal a novel function of plasma membrane-localized Hsp40 protein in modulating, alongside chaperon regulator OsBAG4, transcriptional regulation under salinity stress tolerance.

Callus induction, which results in fate transition in plant cells, is considered as the first and key step for plant regeneration. This process can be stimulated in different tissues by a callus-inducing medium (CIM), which contains a high concentration of phytohormone auxin. Although a few key regulators for callus induction have been identified, the multiple aspects of the regulatory mechanism driven by high levels of auxin still need further investigation. Here, we find that high auxin induces callus through a H3K36 histone methylation-dependent mechanism, which requires the methyltransferase SET DOMAIN GROUP 8 (SDG8). During callus induction, the increased auxin accumulates SDG8 expression through a TIR1/AFBs-based transcriptional regulation. SDG8 then deposits H3K36me3 modifications on the loci of callus-related genes, including a master regulator WOX5 and the cell proliferation-related genes, such as CYCB1.1. This epigenetic regulation in turn is required for the transcriptional activation of these genes during callus formation. These findings suggest that the massive transcriptional reprogramming for cell fate transition by auxin during callus formation requires epigenetic modifications including SDG8-mediated histone H3K36 methylation. Our results provide insight into the coordination between auxin signaling and epigenetic regulation during fundamental processes in plant development.

The mRNA modification N⁶-methyladenosine (m⁶A) plays vital roles in plant development and biotic and abiotic stress responses. The RNA m⁶A demethylase ALKBH9B can remove m⁶A in alfalfa mosaic virus RNA and plays roles in alfalfa mosaic virus infection in Arabidopsis. However, it is unknown whether ALKBH9B also exhibits demethylation activity and has a biological role in endogenous plant mRNA. We demonstrated here that mRNA m⁶A modification is induced by the phytohormone abscisic acid (ABA) and that ALKBH9B has m⁶A demethylation activity on endogenous mRNA. Knocking out ALKBH9B led to hypersensitivity to ABA treatment during seed germination and early seedling development. We further showed that ALKBH9B removes the m⁶A modification in the ABA INSENSITIVE 1 (ABI1) and BRI1-EMS-SUPPRESSOR 1 (BES1) transcripts following ABA treatment, affecting the stability of these mRNAs. Furthermore, we determined that ALKBH9B acts genetically upstream of the transcription factors ABI3 and ABI5, and its regulatory function in ABA responses depended on ABI3 and ABI5. Our findings reveal the important roles of the m⁶A modification in ABA responses and highlight the role of ALKBH9B-mediated m⁶A demethylation in regulating ABA responses post-transcriptionally.