Since the whole-genome sequencing of many crops has been achieved, crop functional genomics studies have reached the large-scale and high-throughput stage. However, phenotypic data acquisition is still a bottleneck restricting crop breeding and functional genomics studies. Now technological advances allow us to relieve the bottleneck and explore more in the coming decades. Thus, in this article, we review main developments on high-throughput phenotyping in the controlled environments and field conditions as well as for post-harvest yield and quality assessment in past decades. Then, we describe the latest multiomics works combining high-throughput phenotyping and genetic studies. Finally, some conceptual challenges and future perspectives are also proposed. We hope to provide useful information and alternative phenotyping solutions for how to bridge the phenotype-genotype gap and believe that such endeavors in high-throughput phenotyping will accelerate plant genetic improvement and promote the next green revolution in crop breeding.

Speciation has long been regarded as an irreversible process once the reproductive barriers had been established. However, unlike in natural populations, artificial selection might either accelerate or prevent speciation processes in domesticated species. Asian cultivated rice is a target crop for both domestication and artificial breeding; it contains two subspecies of indica and japonica, which usually produce sterile inter-subspecific hybrids due to reproductive barriers. Here we constructed the evolutionary trajectory of a reproductive isolation system S5, which regulates fertility in indica-japonica hybrids via three adjacent genes, based on data of 606 accessions including two cultivated and 11 wild rice species. Although hybrid sterility haplotypes at S5 led to establishment of a killer-protector reproductive barrier, origin of wide-compatibility haplotypes by complex hybridization and recombination provided an opposing force to reproductive isolation and thus prevented speciation during domestication. Analysis in a diallel set of 209 crosses involving 21 parents showed that the wide-compatibility genotypes largely reduced sterility of indica-japonica hybrids indicating that wide compatibility gene would enable gene flow to maintain species coherence. This counter-acting system indicates that combined effects of natural evolution and artificial selection may result in reversible processes of speciation in rice, which may also have implications for rice genetic improvement.

Iron (Fe) deficiency is prevalent in plants grown in neutral or alkaline soil. Plants have evolved sophisticated mechanisms that regulate Fe homeostasis, ensuring survival. In Arabidopsis FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) is a crucial regulator of Fe deficiency response. FIT is activated indirectly by bHLH IVc transcription factors (TFs) under Fe deficiency; however, it remains unclear which protein(s) act as a linker to mediate the activation of FIT by bHLH IVc TFs. In this study, we characterize the functions of bHLH121 and demonstrate that it directly associates with the FIT promoter. We found that loss-of-function mutations of bHLH121 cause severe Fe deficiency symptoms, reduced Fe accumulation, and disrupted expression of genes associated with Fe homeostasis. Genetic analysis showed that FIT is epistatic to bHLH121 and FIT overexpression partially rescues the bhlh121 mutant. Further investigation revealed, that bHLH IVc TFs interact with and promote nuclear accumulation of bHLH121. bHLH121 has DNA-binding function and can bind the promoters of FIT and bHLH Ib genes, but we did not find that it has either direct transcriptional activation or repression activity toward FIT and bHLH Ib genes. Meanwhile, we found that bHLH121 functions downstream of and is a direct target of bHLH IVc TFs, and its expression is induced by Fe deficiency in a bHLH IVc-dependent manner. Taken together, these results establish a direct link that bHLH121 functions together with bHLH IVc TFs to mediate the activation of FIT and thus plays a pivotal role in maintaining Fe homeostasis in Arabidopsis.

The phytohormone abscisic acid (ABA) plays pivotal roles in triggering stomatal closure and facilitating adaptation of plants to drought stress. Hydrogen Sulfide (H2S), a small signaling gas molecule, is involved in ABA-dependent stomatal closure. However, how H2S regulates ABA signaling remains largely unclear. Here, we show that ABA induces the production of H2S catalyzed by L-CYSTEINE DESULFHYDRASE1 (DES1) in guard cells and H2S in turn positively regulates ABA signaling through persulfidation of Open Stomata 1 (OST1)/SNF1-RELATED PROTEIN KINASE2.6 (SnRK2.6). Two cysteine (Cys) sites, Cys131 and Cys137 that are exposed on the surface of SnRK2.6 and closed to the activation loop, were identified to be persulfidated, which promotes the activity of SnRK2.6 and its interaction with ABA response element-binding factor2 (ABF2), a transcription factor downstream of ABA signaling. When Cys131, Cys137 or both in SnRK2.6 was substituted with Serine (S), H2S-induced SnRK2.6 activity and SnRK2.6-ABF2 interaction were partially (SnRK2.6C131S and SnRK2.6C137S) or completely (SnRK2.6C131SC137S) comprised. Introduction of SnRK2.6C131S, SnRK2.6C137S, or SnRK2.6C131SC137S into ost1-3 mutant could not rescue the mutant phenotype, showing less sensitive to ABA- and H2S-induced stomatal closure and Ca2+ influx as well as increased water loss and decreased drought tolerance. Taken together, our study reveals a novel post-translational regulatory mechanism of ABA signaling in which H2S persulfidates SnRK2.6 to promote ABA signaling and ABA-induced stomatal closure.

Plants maintain their internal temperature by adjusting their morphology and architecture under environments with fluctuating temperatures, an adaptive process termed thermomorphogenesis. Notably, the rhythmic patterns of plant thermomorphogenesis are governed by daylength information. However, it remains elusive how thermomorphogenic rhythms are regulated by photoperiod. Here, we show that warm temperatures enhance the accumulation of the chaperone GIGANTEA (GI), which thermostabilizes REPRESSOR OF ga1-3 (RGA) under long days, thereby attenuating PHYTOCHROME INTERACTING FACTOR 4 (PIF4)-mediated thermomorphogenesis. In contrast, under short days, when GI accumulation is reduced, RGA is readily degraded through the gibberellic acid (GA)-mediated ubiquitination-proteasome pathway, promoting thermomorphogenic growth. These data indicate that the GI-RGA-PIF4 signaling module enables plant thermomorphogenic responses to occur in a daylength-dependent manner. We propose that the GI-mediated integration of photoperiodic and temperature information shapes thermomorphogenic rhythms, which enable plants to adapt to diel fluctuations in daylength and temperature during seasonal transitions.

Plant and non-plant species possess cryptochrome (CRY) photoreceptors to mediate blue-light regulation of development or the circadian clock. The blue light-dependent homooligomerization of Arabidopsis CRY2 is a known early photoreaction necessary for its functions, but the photobiochemistry and function of light-dependent homooligomerization and heterooligomerization of cryptochromes, collectively referred to as CRY photooligomerization, have not been well-established. Here we show that photooligomerization is an evolutionarily conserved photoreaction characteristic of the CRY photoreceptors in plant and some non-plant species. Our analyses of the kinetics of the forward and reverse reactions of photooligomerization of Arabidopsis CRY1 and CRY2 provide a previously unrecognized mechanism underlying the different photosensitivity and photoreactivity of these two closely related photoreceptors. We found that photooligomerization is necessary but not sufficient for the functions of CRY2, implying that CRY photooligomerization must accompany with additional function-empowering conformational changes. We further demonstrate that the CRY2-CRY1 heterooligomerization plays roles in regulating functions of Arabidopsis CRYs in vivo. These results are consistent with the hypothesis that photooligomerization is an evolutionary conserved mechanism that determines the photosensitivity and photoreactivity of plant CRYs.

Stomatal ontogenesis, patterning, and function are hallmarks of environmental plant adaptation, especially to conditions limiting plant growth such as elevated temperatures and reduced water availability. The specification and distribution of a stomatal cell lineage and its terminal differentiation to guard cells requires a master regulatory protein phosphorylation cascade initiated by the YODA mitogen-activated protein kinase kinase kinase. YODA signaling results in the activation of MITOGEN-ACTIVATED PROTEIN KINASEs (MPK3 and MPK6), which confer regulation of transcription factors including SPEECHLESS (SPCH). Herein, we report that acute heat stress affects the phosphorylation and deactivation of SPCH and modulates stomatal density. By using complementary molecular, genetic, biochemical and cell biology approaches we provide solid evidence that HEAT SHOCK PROTEINS 90 (HSP90) play a crucial role in transducing heat-stress response through YODA cascade. Genetic studies revealed that YODA and HSP90.1 are epistatic, and they likely function linearly in the same developmental pathway regulating stomata formation. HSP90s interact with YODA, affect its cellular polarization, and modulate the phosphorylation of downstream targets, like MPK6 and SPCH, under both normal and heat-stress conditions. Thus, HSP90-mediated specification and differentiation of stomatal cell lineage couples stomatal development to environmental cues providing an adaptive mechanism to plant heat-stress response.

The molecular links between extracellular signals and the regulation of localized protein synthesis in plant cells are poorly understood. Here, we show that in Arabidopsis thaliana, the extracellular peptide RALF1 and its receptor, the FERONIA receptor kinase, promote root hair (RH) tip growth by modulating protein synthesis. The underlying mechanism is that RALF1 promotes the FERONIA-mediated phosphorylation of eIF4E1, a component that plays a crucial role in the control of the mRNA translation rate. Phosphorylated eIF4E1 increases mRNA affinity and modulates mRNA translation and, thus, protein synthesis. The mRNAs targeted by the RALF1-FERONIA-eIF4E1 module include ROP2 and RSL4, which are important for RH cell polarity and growth. RALF1-FERONIA expressed in a polar manner in RHs facilitates eIF4E1 polar localization and may further control local ROP2 translation. Moreover, high levels of RSL4 exert a negative feedback on RALF1 expression via directly binding the RALF1 gene promoter, determining the final RH size. The link between RALF1–FERONIA signaling and protein synthesis constitutes a novel component regulating cell expansion in these growing polar cells.

Post-translational modifications play essential roles in finely modulating eukaryotic circadian clock systems. In plants, the effects of O-glycosylation on the circadian clock and the underlying mechanisms remain largely unknown. O-fucosyltransferase SPINDLY (SPY), and O-GlcNAc transferase SECRET AGENT (SEC) are two prominent O-glycosylation enzymes in higher plants, with both overlapped and unique functions in plant growth and development. Unlike the critical role of O-GlcNAc in regulating animal circadian clock, here we demonstrated that nuclear localized SPY but not SEC, specifically modulates the pace of Arabidopsis circadian clock. By identifying the interactome of SPY, we characterized PSEUDO RESPONSE REGULATOR 5 (PRR5), one of core circadian clock components, as a new SPY-interacting protein. PRR5 can be O-fucosylated by SPY in planta, while point mutations in the SPY catalytic domain abolishes the O-fucosylation of PRR5. The protein abundance of PRR5 is strongly increased in spy mutants, while the degradation rate of PRR5 is much reduced, suggesting PRR5 proteolysis is promoted by SPY-mediated O-fucosylation. Moreover, multiple lines of genetic evidence indicate PRR5 is a major downstream target of SPY to specifically mediate its modulation on circadian clock. Collectively, our findings provide a novel insight into the specific role of O-fucosyltransferase activity of SPY in modulating the circadian clock, and implicate O-glycosylation might have an evolutionarily conserved role in modulating circadian clock system, via O-GlcNAcylation in mammals, but via O-fucosylation in higher plants.

Root-knot nematodes (RKN, genus Meloidogyne) are a class of plant parasites that seek out and infect roots of many plant species. It is believed that RKN target certain signaling molecules derived from plants to locate their hosts, however currently no plant compound has been unambiguously identified as a universal RKN attractant. To address this question, we screened a chemical library of synthetic compound for M. incognita attractants. Break-down product of aminopropylamino-anthraquinone, 1,3-diaminopropane, as well as related compounds putrescine and cadaverine were found to attract M. incognita. After examining various polyamines, M. incognita were found to be attracted specifically by natural compounds that possess three to five methylene groups between two terminal amino groups. Using cryo-TOF-SIMS/SEM, cadaverine was indeed detected in soybean root cortex cells and the surrounding rhizosphere, establishing a chemical gradient. In addition to cadaverine, putrescine and 1,3-diaminopropane were also detected in root exudate by HPLC-MS/MS. Furthermore, exogenously applied cadaverine is sufficient to enhance M. incognita infection of Arabidopsis seedlings. These results suggest M. incognita may indeed target polyamines to locate the appropriate host plants, and these naturally-occurring polyamines may have viable applications in agriculture to develop protection strategies for crops from RKN infections.

Epigenetic regulation of gene expression is important for plant adaptation to environmental changes. Previous results showed that Arabidopsis RPD3-like histone deacetylase HDA9 has a function to repress plant response to stress. The underling mechanism by which this epigenetic regulator targets to specific chromatin loci to control gene expression networks involved in plant response to stress remains unknown. Here, we show that HDA9 represses stress-tolerance by interacting with and regulating DNA-binding and transcriptional activity of WRKY53 that functions as a higher hierarchy positive regulator of stress resistance. We found that WRKY53 is post-translationally modified by lysine acetylation at multiple sites, some of which are removed by HDA9 resulting in inhibition of WRKY53 transcription activity. Conversely, WRKY53 displays a negative function on the HDA9 histone deacetylase activity. The results indicate that HDA9 and WRK53 are reciprocal negative regulators of their activities and reveal functional interplay between a chromatin regulator and a transcription factor to regulate stress tolerance in plants.

Salicylic acid (SA) has long been known to be essential for basal defense and systemic acquired resistance (SAR). N-hydroxypipecolic acid (NHP), a recently discovered plant metabolite, also plays a key role in SAR and to a less extent in basal resistance. Following pathogen infection, levels of both compounds are dramatically increased. Analysis of SA- or SAR-deficient mutants has uncovered how SA and NHP are biosynthesized. The completion of the SA and NHP biosynthetic pathways in Arabidopsis allowed better understanding of how they are regulated. This review discusses recent progresses on SA and NHP biosynthesis and their regulation in plant immunity.

Advances in the detection and mapping of messenger RNA (mRNA) N6-methyladenosine (m6A) and 5-methylcytosine (m5C) and DNA N6-methyldeoxyadenosine (6mA) redefined our understanding of these modifications as additional tiers of epigenetic regulation. In plants, the most prevalent internal mRNA modifications, m6A and m5C, play crucial and dynamic roles in many processes, including embryo development, stem cell fate determination, trichome branching, leaf morphogenesis, floral transition, stress responses, fruit ripening and root development. The newly identified and widespread epigenetic marker 6mA DNA methylation is associated with gene expression, plant development and stress responses. Here, we review the latest progress in the research on mRNA and DNA epigenetic modifications, including the detection, dynamics, distribution, functions, regulatory proteins and evolution, with a focus on m6A, m5C and 6mA, and discuss future perspectives in plants.

Rice tillering, a key architecture trait to determine grain yield, is highly regulated by a class of newly identified phytohormone, strigolactones (SLs). However, their whole signaling pathway from receptor to downstream transcription factor to finally inhibit tillering remains unrevealed. We first found that brassinosteroids (BRs) strongly enhanced tillering by promoting bud outgrowth in rice, which is largely different from that in Arabidopsis. Genetic and biochemical analysis indicate that both SL and BR signaling controls rice tillering through regulating the stability of D53 and/or OsBZR1–RLA1–DLT module, the transcriptional complex in rice BR signaling pathway. Further experiments demonstrate that D53 interacts with OsBZR1 to inhibit FC1 expression, the local inhibitor for tillering, which depends on the direct DNA-binding by OsBZR1 to recruit D53 to FC1 promoter in rice buds. Thus, these findings uncover the mechanism how SLs and BRs coordinately regulate rice tillering via the early responsive gene FC1.

The rubber tree, Hevea brasiliensis, produces natural rubber that serves as an essential industrial raw material. Here, we present a high-quality reference genome for a rubber tree cultivar GT1 using single-molecule real-time sequencing (SMRT) and Hi-C technologies to anchor the ∼1.47-Gb genome assembly into 18 pseudo-chromosomes. The chromosome-based genome analysis enabled us to establish a model of spurge chromosome evolution since the common paleopolyploid event occurred before the split of Hevea and Manihot. We show recent and rapid bursts of the three Hevea-specific LTR-retrotransposon families during the last ten million years (MYR), leading to the massive expansion ∼60.44% (∼890 Mbp) of the whole rubber tree genome since the divergence from Manihot. We identify large-scale expansion of genes associated with whole rubber biosynthesis processes, such as basal metabolic processes, ethylene biosynthesis, and the activation of polysaccharide and glycoprotein lectin, which are important properties for the latex production. We report the first map of genomic variation between the cultivated and wild rubber trees and obtained ∼15.7 million high-quality single nucleotide polymorphisms (SNPs). We identified hundreds of candidate domestication genes with drastically lowered genomic diversity in the cultivated but not wild rubber trees despite a relatively short domestication history, some of which are involved in the rubber biosynthesis. This genome assembly represents key resources for future rubber tree breeding programs providing novel gene targets to improve biotic and abiotic tolerance and rubber production.

In plants, RNA interference (RNAi) plays a pivotal role in growth and development, and responses to environmental inputs, including pathogen attack. The intercellular and systemic trafficking of small interfering/micro(si/mi)RNA is a central component in this regulatory pathway. Currently, little is known with regards to the molecular agents involved in the movement of these si/miRNAs. To address this situation, we employed a biochemical approach to identify and characterize a conserved SMALL RNA-BINDING PROTEIN 1 (SRBP1) family that mediates non-cell-autonomous small (s)RNA trafficking. In Arabidopsis, AtSRBP1 is a glycine-rich RNA-binding protein, also known as AtGRP7, which we show binds single-stranded-siRNA. A viral vector, Zucchini yellow mosaic virus (ZYMV), was employed to functionally characterized the AtSRBP1-4 (AtGRP7/2/4/8) RNA recognition motif (RRM) and GR domains. Cellular-based studies revealed the GR domain as being necessary and sufficient for SRBP1 cell-to-cell movement. Our findings provide a foundation for future research into the mechanism and function of mobile sRNA signaling agents in plants.

Phytochromes are red/far-red reversible photoreceptors essential for plant growth and development. Phytochrome signaling is mediated by the physiologically active far-red-absorbing Pfr form that can be inactivated to the red-absorbing Pr ground state by light dependent photoconversion or by light independent thermal reversion also termed dark reversion. Although the term “dark reversion” is justified by historical reasons and frequently used in the literature, “thermal reversion” is more appropriately describing the process of light-independent but temperature regulated Pfr relaxation that not only occurs in darkness but also in light and will be used throughout the review. Thermal reversion is a critical parameter for the light sensitivity of phytochrome mediated responses and has been studied for decades often resulting in contradictory findings. Thermal reversion is an intrinsic property of the phytochrome molecule but can be modulated by intra- and intermolecular interactions as well as biochemical modifications such as phosphorylation. In this review, we outline the history of phytochrome thermal reversion studies highlighting important predictions that have been made before knowing the molecular basis. We further summarize recent findings about the molecular mechanisms regulating phytochrome thermal reversion and its functional role in light and temperature sensing in plants.

Cuscuta species (dodders) are holoparasites that totally rely on host plants to survive. Although various mobile proteins have been identified to travel within a plant, whether and to what extent protein transfer between Cuscuta and host plants remain unclear. We found that hundreds to more than 1500 proteins were transferred between Cuscuta and the host plants Arabidopsis and soybean, and hundreds of inter-plant mobile proteins were even detected in the seeds of Cuscuta and the host soybean. Different hosts bridge-connected by dodder were also found to exchange hundreds of proteins. Quantitatively, the mobile proteins represent a few to more than 10% of the proteomes of the foreign plants. Using Arabidopsis plants expressing different reporter proteins, we show that these reporter proteins could travel between plants, and importantly, retained their activity in the foreign plants. Comparison between the inter-plant mobile proteins and mRNAs indicated that the majority of the mobile proteins were not de novo synthesized from the translocated mRNAs, but bona fide mobile proteins. We propose that large-scale inter-plant protein translocation may play an important role in the interactions between host plants and dodder and even among the dodder bridge connected hosts.

In plants, high disease resistance often results in a fitness penalty to plant growth. Therefore, breeding crops with a balanced yield and disease resistance has become a major challenge. Recently, microRNA (miRNA)-mediated R gene turnover has been shown to be a protective mechanism for plants to prevent autoimmunity in the absence of pathogens. However, whether these miRNAs play a role in plant growth and how miRNA-mediated R gene turnover responds to pathogen infection have been rarely explored. Here, we identified the Brassica miRNA, miR1885, targets both immune receptor gene and development-related gene for negative regulation through distinct modes of action. MiR1885 directly silences a TIR-NBS-LRR class of R gene BraTNL1 but represses the expression of photosynthesis-related gene BraCP24 through Trans-Acting Silencing (TAS) gene BraTIR1-mediated silencing. We found that, under natural conditions, miR1885 was kept in low levels to maintain normal development and basal immunity but peaked during the floral transition to promote flowering. Interestingly, upon Turnip mosaic virus (TuMV) infection, miR1885-dependent trans-acting silencing of BraCP24 was enhanced to speed up floral transition, whereas miR1885-mediated R gene turnover was overwhelmed by TuMV-induced BraTNL1 expression, reflecting an integrative regulation of the arms race between plants and pathogens. Collectively, our results demonstrate that a single Brassica miRNA dynamically regulates both innate immunity and plant growth and responds to viral infection, therefore demonstrating an integrative strategy for Brassica in modulating the interplay between growth, immunity and pathogen infection.

Coix is a grass crop domesticated as early as Neolithic era. It is still widely cultivated for both highly nutritional food and medicinal use. However, the genetic study and breeding of this crop are hindered by lack of a sequenced genome. Here we report de novo sequencing and assembly of the 1,619 Mb genome of Coix, and annotation of 75.39% repeats and 39,629 protein-coding genes. Comparative genomics analysis showed that Coix is more closely related to sorghum rather than maize, but intriguingly only Coix and maize had a recent whole genome duplication event which was not detected in sorghum. We further constructed a genetic map and mapped several important traits especially the strength of hull. Selection of papery hull (thin, easy-dehulling) from the stony one (thick, hard-dehulling) in wild progenitors was a key step in Coix domestication. The papery hull makes seed easier to process and germinate. Anatomic and global transcriptome analysis revealed that the papery hull is resulted from inhibition of cell division and wall biogenesis. We also successfully demonstrated that seed hull pressure resistance is controlled by two major quantitative trait loci (QTLs), which are associated with hull thickness and color, respectively. The two QTLs were further fine mapped within intervals of 250 Kb and 146 Kb, respectively. These resources provide a platform for evolutionary studies and will facilitate molecular breeding of this important crop.

Coix lacryma-jobi L., a plant species closely related to Zea and Sorghum, is an important food and medicinal crop in Asia. However, no reference genome of this species has been reported, and its exact phylogeny within the Andropogoneae remains unresolved. Here, we generated a high-quality genome assembly of coix comprising ∼1.73 Gb with 44,485 predicted protein-coding genes. We found the coix is a typical diploid plant with an overall 1-to-1 syntenic relationship with the sorghum genome, despite its drastic genome expansion (∼2.3-fold) due mainly to the activity of transposable elements. Phylogenetic analysis revealed that coix diverged with sorghum ∼10.41 million years ago, which was ∼1.49 million years later than the divergence between sorghum and maize. Resequencing of 27 additional coix accessions revealed that they could be unambiguously separated into wild relatives and cultivars, and coix experienced a strong genetic bottleneck which lost about half of the genetic diversity during domestication, even though many traits have remained undomesticated. Our data not only provide novel comparative genomic and evolutionary insights into the Andropogoneae lineage, but also an important resource that will greatly benefit the molecular breeding of this important crop.

Salicylic acid (SA) is an important phytohormone mediating both local and systemic defense responses in plants. Despite over half a century of research, how plants biosynthesize SA remains unresolved. In Arabidopsis, a major part of SA is derived from isochorismate, a key intermediate produced by the isochorismate synthase (ICS), which is reminiscent of SA biosynthesis in bacteria. Whereas bacteria employ an isochorismate pyruvate lyase (IPL) that catalyzes the turnover of isochorismate to pyruvate and SA, plants do not contain an IPL ortholog and generate SA from isochorismate through an unknown mechanism. Combining genetic and biochemical approaches, we delineated the SA biosynthetic pathway downstream of isochorismate in Arabidopsis. We show that PBS3, a GH3 acyl adenylase-family enzyme important for SA accumulation, catalyzes ATP- and Mg2+-dependent conjugation of L-glutamate primarily to the 8-carboxyl of isochorismate and yields the key SA biosynthetic intermediate isochorismoyl-glutamate A. Moreover, EPS1, a BAHD acyltransferase-family protein with previously implicated role in SA accumulation upon pathogen attack, harbors a noncanonical active site and an unprecedented isochorismoyl-glutamate A pyruvoyl-glutamate lyase (IPGL) activity that produces SA from the isochorismoyl-glutamate A substrate. Together, PBS3 and EPS1 form a two-step metabolic pathway to produce SA from isochorismate in Arabidopsis, which is distinct from how SA is biosynthesized in bacteria. This study closes a major knowledge gap in plant SA metabolism and would help develop new strategies for engineering disease resistance in crop plants.

Precursor mRNA (pre-mRNA) splicing is essential for gene expression in most eukaryotic organisms. Previous studies from mammals, Drosophila and yeast show that the majority of splicing events occurs co-transcriptionally. In plants, however, the nature of co-transcriptional splicing (CTS) and its regulation are still largely unknown. Here, we used chromatin-bound RNA sequencing (CB-RNA-seq), to study CTS in Arabidopsis thaliana. CTS was widespread in Arabidopsis seedlings, with a large proportion of alternative splicing events determined co-transcriptionally. CTS efficiency correlated with gene expression level, the chromatin landscape and, most surprisingly, the number of introns and exons of individual genes, but was independent of gene length. In combination with eCLIP-seq analysis, our results showed that the hnRNP-like proteins RZ-1B and RZ-1C promote efficient CTS globally through direct binding, frequently to exonic sequences. Notably, this general effect of RZ-1B/1C on splicing promotion was mainly at the chromatin level, not at the mRNA level. RZ-1C promotes CTS of multiple exons genes in association with its binding to the regions both proximal and distal to the regulated introns. We propose that RZ-1C promotes efficient CTS of genes with multiple exons involving cooperative interactions with many exons, introns and splicing factors. Our work thus reveals important features of CTS in plants and provides methodologies for the investigation of CTS and RNA-binding proteins in plants.

The RNA-binding pentatricopeptide repeat (PPR) family comprises hundreds to thousands of genes in most plants, but only a few dozen in algae, evidence of massive gene expansions during land plant evolution. The nature and timing of these expansions has not been well-defined due to the sparse sequence data available from early-diverging land plant lineages. We exploit the comprehensive OneKP dataset of over 1,000 transcriptomes from diverse plants and algae to establish a clear picture of the evolution of this massive gene family, focusing on the proteins typically associated with RNA editing, which show the most spectacular variation in numbers and domain composition across the plant kingdom. We characterise over 2,250,000 PPR motifs in over 400,000 proteins. In lycophytes, polypod ferns and hornworts, nearly 10% of expressed protein-coding genes encode putative PPR editing factors, whereas they are absent from algae and complex-thalloid liverworts. We show that rather than a single expansion, most land plant lineages with high numbers of editing factors have continued to generate novel sequence diversity. We identify sequence variation that implies functional differences between PPR proteins in seed plants versus non-seed plants and which we propose to be linked to seed-plant-specific editing cofactors. Finally, using the sequence variation across the dataset, we develop a structural model of the catalytic DYW domain associated with C-to-U editing and identify a clade of unique DYW variants that are strong candidates as U-to-C RNA editing factors, given their phylogenetic distribution and sequence characteristics.

RNA splicing and spliceosome assembly in eukaryotes occur mainly during transcription. However, co-transcriptional splicing has not yet been explored in plants. Here, we built nascent transcriptomes of chromatin RNAs in Arabidopsis thaliana and showed that, nearly all introns undergo co-transcriptional splicing, and this occurs with higher efficiency for introns in protein-coding genes than for noncoding RNAs. Total intron number and intron position are two predominant features that correlate with co-transcriptional splicing efficiency, and introns with alternative 5′ or 3′ splice sites are less efficiently spliced. Furthermore, we found that trans-acting protein mutations lead to more introns with increased splicing defects in nascent RNAs than in mature RNAs, and that introns with increased splicing defects in mature RNAs are inefficiently spliced at the co-transcriptional level. Our results not only uncovered widespread co-transcriptional splicing in Arabidopsis, but also identified features that may affect or be affected by co-transcriptional splicing efficiency.

In the phloem cap region of Arabidopsis plants, sulfur-rich cells (S-cells) accumulate >100 mM glucosinolates (GLS), but are biosynthetically inactive. The source and route of S-cell-bound GLS remain elusive. In this study, using single-cell sampling and scanning electron microscopy with energy-dispersive X-ray analysis we show that two GLS importers, NPF2.10/GTR1 and NPF2.11/GTR2, are critical for GLS accumulation in S-cells, although they are not localized in the S-cells. Comparison of GLS levels in S-cells in multiple combinations of homo- and heterografts of gtr1 gtr2, biosynthetic null mutant and wild-type plants indicate that S-cells accumulate GLS via symplasmic connections either directly from neighboring biosynthetic cells or indirectly to non-neighboring cells expressing GTR1/2. Distinct sources and transport routes exist for different types of GLS, and vary depending on the position of S-cells in the inflorescence stem. Based on these findings, we propose a model illustrating the GLS transport routes either directly from biosynthetic cells or via GTR-mediated import from apoplastic space radially into a symplasmic domain, wherein the S-cells are the ultimate sink. Similarly, we observed accumulation of the cyanogenic glucoside defensive compounds in high-turgor cells in the phloem cap of Lotus japonicus, suggesting that storage of defensive compounds in high-turgor cells may be a general mechanism for chemical protection of the phloem cap.

WD40 repeat-containing proteins (WD40 proteins) serve as versatile scaffolds for protein–protein interactions, modulating a variety of cellular processes such as plant stress and hormone responses. Here we report the identification of a WD40 protein, XIW1 (for XPO1-interacting WD40 protein 1), which positively regulates the abscisic acid (ABA) response in Arabidopsis. XIW1 is located in the cytoplasm and nucleus. We found that it interacts with the nuclear transport receptor XPO1 and is exported by XPO1 from the nucleus. Mutation of XIW1 reduces the induction of ABA-responsive genes and the accumulation of ABA Insensitive 5 (ABI5), causing mutant plants with ABA-insensitive phenotypes during seed germination and seedling growth, and decreased drought stress resistance. ABA treatment upregulates the expression of XIW1, and both ABA and abiotic stresses promote XIW1 accumulation in the nucleus, where it interacts with ABI5. Loss of XIW1 function results in rapid proteasomal degradation of ABI5. Taken together, these findings suggest that XIW1 is a nucleocytoplasmic shuttling protein and plays a positive role in ABA responses by interacting with and maintaining the stability of ABI5 in the nucleus.

The Arabidopsis thaliana Calmodulin-binding Transcription Activator (CAMTA) transcription factors CAMTA1, CAMTA2 and CAMTA3 (CAMTA123) serve as master regulators of salicylic acid (SA)-mediated immunity, repressing the biosynthesis of SA in healthy plants. Here we show that CAMTA123 also repress the biosynthesis of pipecolic acid (Pip) in healthy plants. Loss of CAMTA123 function resulted in the induction of AGD2-like Defense Response Protein 1 (ALD1), which encodes an enzyme involved in Pip biosynthesis. Induction of ALD1 resulted in the accumulation of high levels of Pip which brought about increased levels of the SA-receptor protein NPR1 without induction of NPR1 or requirement for an increase in SA levels. Pip-mediated induction of ALD1 and genes regulating the biosynthesis of SA—CBP60g, SARD1, PAD4 and EDS1—was largely dependent on NPR1. Further, Pip-mediated increase in NPR1 protein levels was associated with priming of Pip and SA biosynthesis genes to induction by low levels of SA. Taken together, our findings expand the role for CAMTA123 in regulating key immunity genes and suggest a working model whereby loss of CAMTA123 repression leads to the induction of plant defense genes and initiation of SAR.

Two signal molecules, salicylic acid (SA) and N-hydroxypipecolic acid (NHP), play critical roles in plant immunity. The biosynthetic genes of both compounds are positively regulated by master immune-regulating transcription factors SARD1 and CBP60g. However, the relationship between the SA and NHP pathways is unclear. On the other hand, CALMODULIN BINDING TRANSCRIPTION FACTOR 1 (CAMTA1), CAMTA2 and CAMTA3 are redundant negative regulators of plant immunity, the mechanism of which remains unknown. Here, through chromatin immunoprecipitation and electrophoretic mobility shift assays, we uncovered that CBP60g is a direct target of CAMTA3. The autoimmunity of camta3-1 is suppressed by sard1 cbp60g double mutant as well as ald1 and fmo1, two mutants defective in NHP biosynthesis. Interestingly, a suppressor screen using the camta1/2/3 triple mutant yielded various mutants blocking biosynthesis or signaling of either SA or NHP, leading to nearly complete suppression of the extreme autoimmunity of camta1/2/3, suggesting that the SA and NHP pathways can mutually amplify each other. Together, these results reveal that the CAMTAs repress the biosynthesis of SA and NHP by modulating the expression of SARD1 and CBP60g, and that the SA and NHP pathways are coordinated to optimize plant immune response.

Symbiotic microorganisms improve nutrient uptake by plants. To initiate mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, plants perceive Myc factors, including lipochitooligosaccharides (LCOs) and short-chain chitooligosaccharides (CO4/CO5), secreted by AM fungi. However, the molecular mechanism of Myc factors perception remains elusive. Here, we identified a heteromer of LysM receptor-like kinases, OsMYR1/OsLYK2 and OsCERK1, that mediates perception of AM fungi in rice. CO4 directly binds to OsMYR1, promoting the dimerization and phosphorylation of this receptor complex. Compared to control plants, Osmyr1 and Oscerk1 mutant rice plants are less sensitive to Myc factors and show decreased AM colonization. We engineered transgenic rice by expressing chimeric receptors that respectively replaced the ectodomains of OsMYR1 and OsCERK1 with those from the homologous Nod factor receptors MtNFP and MtLYK3 of Medicago truncatula. Transgenic plants displayed increased calcium oscillations in response to Nod factors compared to control rice. Our findings reveal a mechanism for mycorrhizal symbiotic signal perception in rice, and the ectopic expression of chimeric Nod/Myc receptors achieves a potentially important step towards generating cereals that host nitrogen-fixing bacteria.

Suppression mechanisms employed by transcriptional repressors commonly exist in diverse phytohormone signaling pathways. In Arabidopsis thaliana, JASMONATE-ZIM DOMAIN (JAZ) proteins are transcriptional repressors that function as negative regulators of diverse JA responses. Novel Interactor of JAZ (NINJA) is an adaptor protein connecting JAZs with the co-repressor, TOPLESS (TPL), to mediate gene repression in JA-dependent root growth inhibition and defense pathways. However, whether NINJA or other adaptor proteins are employed in other JA responsive biological processes remains to be elucidated. In the present study, we demonstrate that a previously uncharacterized protein, ECAP (EAR motif-Containing Adaptor Protein), directly interacts with JAZ6/8 and enhances their transcriptional repression activities. We also provide evidence that ECAP is a novel adaptor protein for JAZ6/8 recruitment of the transcriptional co-repressor, TPR2, into a transcriptional repressor complex, to repress the WD-repeat/bHLH/MYB complex, an important transcriptional activator in the JA-dependent anthocyanin biosynthesis pathway. This novel insight, together with previous studies, reveals that specific adaptor proteins are critical for distinct JA responses by pairing different JAZs (which possess overlapping but also individual functions) with the general co-repressors, TPL and TPRs.

Plant plasmodesmata (PDs) are specialized channels that enable communication between neighboring cells. The intercellular permeability of PDs affecting plant development, defense and responses to stimulus must be tightly regulated. Analysis of specific PD membrane lipid composition and their impact on PD permeability will provide new insights into PD regulatory mechanism. Herein, we report that the Arabidopsis sld1 sld2 double mutant, lacking sphingolipid long-chain base 8 desaturases 1 and 2, displayed decreased PD permeability due to a significant increase in callose accumulation. Plasmodesmata-located protein 5 (PDLP5) was significantly enriched in the leaf epidermal cells of sld1 sld2 and showed specific binding affinity to phytosphinganine (t18:0), suggesting that the enrichment of t18:0-based sphingolipids in sld1 sld2 PDs might facilitate the recruitment of more PDLP5. The double mutants showed enhanced resistance to the fungal-wilt pathogen Verticillium dahlia or the bacterium Peudomonas syringae pv tomato DC3000. This phenotype was fully restored in sld1 sld2 pdlp5. Thus, we proposed that phytosphinganine might regulate PDs functions and cell-to-cell communication by modifying the level of PDLP5 in PD membranes.

Dietary anthocyanins are important health-promoting antioxidants that make a major contribution to the quality of fruits. It is intriguing that most tomato cultivars do not produce anthocyanins in fruit. However, the purple tomato variety Indigo Rose, which combines the dominant Aft locus and the recessive atv locus from wild tomato species, exhibits light-dependent anthocyanin accumulation in the skin. Here, we report that whereas Aft encodes a functional allele of an anthocyanin activator named SlAN2-like, atv encodes a non-functional allele of the anthocyanin repressor SlMYBATV. The expression of SlAN2-like is responsive to light and a functional SlAN2-like can activate both anthocyanin biosynthetic genes and their regulatory genes, suggesting that SlAN2-like acts as a master regulator and plays a critical role for the activation of anthocyanin biosynthesis. Our results reveal that cultivated tomatoes contain a non-functional allele of this master regulator and therefore fail to produce anthocyanins. Indeed, expression of a functional SlAN2-like in a tomato cultivar led to the activation of the entire anthocyanin biosynthesis pathway and high levels of anthocyanin accumulation in both peel and flesh. Our study exemplifies that efficient engineering of complex metabolic pathways could be achieved through tissue-specific expression of master transcriptional regulators.

The shift of dark-grown seedlings into light causes enormous transcriptome changes followed by a dramatic developmental transition. Here, we show that miRNA biogenesis also undergoes regulatory changes during de-etiolation. Etiolated seedlings maintain low levels of primary-miRNAs (pri-miRNAs) and miRNA processing core proteins, such as Dicer-like 1 (DCL1), SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1), whereas during de-etiolation, both pri-miRNAs and the processing components accumulated to high levels. However, most miRNA levels did not notably increase in response to light. To reconcile this inconsistency, we demonstrate that an unknown suppressor decreases miRNA-processing activity and light-induced SMALL RNA DEGRADING NUCLEASE 1 (SDN1) shortens the half-life of several miRNAs in de-etiolated seedlings. Taken together, we suggest a novel mechanism, miRNA-biogenetic inconsistency, which accounts for the intricacy of miRNA biogenesis during de-etiolation. This mechanism is essential for the survival of de-etiolated seedlings after long-term skotomorphogenesis and their optimal adaptation to ever-changing light conditions.

Ancient whole-genome duplications (WGD or polyploidy) are prevalent in plants, and some WGDs occurred during the timing of severe global environmental changes. It has been suggested that WGDs may have contributed to plant adaptation. However, it still lacks of empirical evidence from genetic level to support the hypothesis. Here, we investigated the survivors of gene duplicates from multiple ancient WGD events on the major branches of angiosperm phylogeny, and aimed to explore genetic evidence supporting the significance of polyploidy. Duplicated genes co-retained from three waves of independent WGDs (∼120 million years ago (Ma), ∼66 Ma and <20 Ma) were investigated in 25 selected species. Gene families functioning in low temperature and darkness were commonly retained gene duplicates after the eight independently occurred WGDs in many lineages around the Cretaceous–Paleocene (K-Pg) boundary, when the global cooling and darkness were the two main stresses. Moreover, the commonly retained duplicates could be key factors which may have contributed to the robustness of the critical stress related pathways. In addition, genome-wide transcription factors (TFs) functioning in stresses tend to retain duplicates after waves of WGDs, and the co-selected gene duplicates in many lineages may play critical roles during severe environmental stresses. Finally, our results shed new light on the significant contribution of paleopolyploidy to plant adaptation during global environmental changes in the evolutionary history of angiosperms.