The organization of the microtubule cytoskeleton is critical for cell and organ morphogenesis. The evolutionarily conserved microtubule-severing enzyme KATANIN plays critical roles in microtubule organization in the plant and animal kingdoms. We previously used conical cell of Arabidopsis thaliana petals as a model system to investigate cortical microtubule organization and cell morphogenesis and determined that KATANIN promotes the formation of circumferential cortical microtubule arrays in conical cells. Here, we demonstrate that the conserved protein phosphatase PP2A interacts with and dephosphorylates KATANIN to promote the formation of circumferential cortical microtubule arrays in conical cells. KATANIN undergoes cycles of phosphorylation and dephosphorylation. Using co-immunoprecipitation coupled with mass spectrometry, we identified PP2A subunits as KATANIN-interacting proteins. Further biochemical studies showed that PP2A interacts with and dephosphorylates KATANIN to stabilize its cellular abundance. Similar to the katanin mutant, mutants for genes encoding PP2A subunits showed disordered cortical microtubule arrays and defective conical cell shape. Taken together, these findings identify PP2A as a regulator of conical cell shape and suggest that PP2A mediates KATANIN phospho-regulation during plant cell morphogenesis.
Flowering time is a fundamental factor determining the global distribution and final yield of rice (Oryza sativa). Although diverse flowering time genes have been reported in this crop, the transcriptional regulation of its key flowering genes are poorly understood. Here, we report that a basic leucine zipper transcription factor, bZIP71, functions as a flowering repressor. The overexpression of bZIP71 delays flowering, while the bzip71 mutant flowers early in both long-day and short-day conditions. A genetic analysis showed that the regulation of flowering by bZIP71 might be independent of Heading date 2 (Hd2), Hd4, and Hd5. Importantly, bZIP71 directly associates with the Early heading date 1 (Ehd1) promoter and represses its transcription, and genetically the function of bZIP71 is impaired in the ehd1 mutant. Moreover, bZIP71 interacts with major components of polycomb repressive complex 2 (PRC2), SET domain group protein 711 (SDG711), and Fertilization independent endosperm 2 (FIE2), through which bZIP71 regulates the H3K27me3 level of Ehd1. Taken together, we present a transcriptional regulatory mechanism in which bZIP71 enhances the H3K27me3 level of Ehd1 and transcriptionally represses its expression, which not only offers a novel insight into a flowering pathway, but also provides a valuable putative target for the genetic engineering and breeding of elite rice cultivars.
Current gene delivery methods for maize are limited to specific genotypes and depend on time-consuming and labor-intensive tissue culture techniques. Here, we report a new method to transfect maize that is culture-free and genotype independent. To enhance efficiency of DNA entry and maintain high pollen viability of 32%-55%, transfection was performed at cool temperature using pollen pretreated to open the germination aperture (40%–55%). Magnetic nanoparticles (MNPs) coated with DNA encoding either red fluorescent protein (RFP), β-glucuronidase gene (GUS), enhanced green fluorescent protein (EGFP) or bialaphos resistance (bar) was delivered into pollen grains, and female florets of maize inbred lines were pollinated. Red fluorescence was detected in 22% transfected pollen grains, and GUS stained 55% embryos at 18 d after pollination. Green fluorescence was detected in both silk filaments and immature kernels. The T1 generation of six inbred lines showed considerable EGFP or GUS transcripts (29%–74%) quantitated by polymerase chain reaction, and 5%–16% of the T1 seedlings showed immunologically active EGFP or GUS protein. Moreover, 1.41% of the bar transfected T1 plants were glufosinate resistant, and heritable bar gene was integrated into the maize genome effectively as verified by DNA hybridization. These results demonstrate that exogenous DNA could be delivered efficiently into elite maize inbred lines recalcitrant to tissue culture-mediated transformation and expressed normally through our genotype-independent pollen transfection system.
Crop breeding during the Green Revolution resulted in high yields largely due to the creation of plants with semi-dwarf architectures that could tolerate high-density planting. Although semi-dwarf varieties have been developed in rice, wheat and maize, none was reported in soybean (Glycine max), and few genes controlling plant architecture have been characterized in soybean. Here, we demonstrate that the auxin efflux transporter PINFORMED1 (GmPIN1), which determines polar auxin transport, regulates the leaf petiole angle in soybean. CRISPR-Cas9-induced Gmpin1abc and Gmpin1bc multiple mutants displayed a compact architecture with a smaller petiole angle than wild-type plants. GmPIN1 transcripts and auxin were distributed asymmetrically in the petiole base, with high levels of GmPIN1a/c transcript and auxin in the lower cells, which resulted in asymmetric cell expansion. By contrast, the (iso)flavonoid content was greater in the upper petiole cells than in the lower cells. Our results suggest that (iso)flavonoids inhibit GmPIN1a/c expression to regulate the petiole angle. Overall, our study demonstrates that a signal cascade that integrates (iso)flavonoid biosynthesis, GmPIN1a/c expression, auxin accumulation, and cell expansion in an asymmetric manner creates a desirable petiole curvature in soybean. This study provides a genetic resource for improving soybean plant architecture.
Drought is a major abiotic stress that limits plant growth and development. Adaptive mechanisms have evolved to mitigate drought stress, including the capacity to adjust water loss rate and to modify the morphology and structure of the epidermis. Here, we show that the expression of CmNF-YB8, encoding a nuclear factor Y (NF-Y) B-type subunit, is lower under drought conditions in chrysanthemum (Chrysanthemum morifolium). Transgenic chrysanthemum lines in which transcript levels of CmNF-YB8 were reduced by RNA interference (CmNF-YB8-RNAi) exhibited enhanced drought resistance relative to control lines, whereas lines overexpressing CmNF-YB8 (CmNF-YB8-OX) were less tolerant to drought. Compared to wild type (WT), CmNF-YB8-RNAi plants showed reduced stomatal opening and a thicker epidermal cuticle that correlated with their water loss rate. We also identified genes involved in stomatal adjustment (CBL-interacting protein kinase 6, CmCIPK6) and cuticle biosynthesis (CmSHN3) that are more highly expressed in CmNF-YB8-RNAi lines than in WT, CmCIPK6 being a direct downstream target of CmNF-YB8. Virus-induced gene silencing of CmCIPK6 or CmSHN3 in the CmNF-YB8-RNAi background abolished the effects of CmNF-YB8-RNAi on stomatal closure and cuticle deposition, respectively. CmNF-YB8 thus regulates CmCIPK6 and CmSHN3 expression to alter stomatal movement and cuticle thickness in the leaf epidermis, thereby affecting drought resistance.
Ammonium (NH4+) and nitrate (NO3−) are major inorganic nitrogen (N) sources for plants. When serving as the sole or dominant N supply, NH4+ often causes root inhibition and shoot chlorosis in plants, known as ammonium toxicity. NO3− usually causes no toxicity and can mitigate ammonium toxicity even at low concentrations, referred to as nitrate-dependent alleviation of ammonium toxicity. Our previous studies indicated a NO3− efflux channel SLAH3 is involved in this process. However, whether additional components contribute to NO3−-mediated NH4+ detoxification is unknown. Previously, mutations in NO3− transporter NRT1.1 were shown to cause enhanced resistance to high concentrations of NH4+. Whereas, in this study, we found when the high-NH4+ medium was supplemented with low concentrations of NO3−, nrt1.1 mutant plants showed hyper-sensitive phenotype instead. Furthermore, mutation in NRT1.1 caused enhanced medium acidification under high-NH4+/low-NO3− condition, suggesting NRT1.1 regulates ammonium toxicity by facilitating H+ uptake. Moreover, NRT1.1 was shown to interact with SLAH3 to form a transporter-channel complex. Interestingly, SLAH3 appeared to affect NO3− influx while NRT1.1 influenced NO3− efflux, suggesting NRT1.1 and SLAH3 regulate each other at protein and/or gene expression levels. Our study thus revealed NRT1.1 and SLAH3 form a functional unit to regulate nitrate-dependent alleviation of ammonium toxicity through regulating NO3− transport and balancing rhizosphere acidification.
Japonica/geng and indica/xian are two major rice (Oryza sativa) subspecies with multiple divergent traits, but how these traits are related and interact within each subspecies remains elusive. Brassinosteroids (BRs) are a class of steroid phytohormones that modulate many important agronomic traits in rice. Here, using different physiological assays, we revealed that japonica rice exhibits an overall lower BR sensitivity than indica. Extensive screening of BR signaling genes led to the identification of a set of genes distributed throughout the primary BR signaling pathway with divergent polymorphisms. Among these, we demonstrate that the C38/T variant in BR Signaling Kinase2 (OsBSK2), causing the amino acid change P13L, plays a central role in mediating differential BR signaling in japonica and indica rice. OsBSK2L13 in indica plays a greater role in BR signaling than OsBSK2P13 in japonica by affecting the auto-binding and protein accumulation of OsBSK2. Finally, we determined that OsBSK2 is involved in a number of divergent traits in japonica relative to indica rice, including grain shape, tiller number, cold adaptation, and nitrogen-use efficiency. Our study suggests that the natural variation in OsBSK2 plays a key role in the divergence of BR signaling, which underlies multiple divergent traits between japonica and indica.
Southern corn leaf blight (SCLB), caused by Bipolaris maydis, is one of the most devastating diseases affecting maize production. However, only one SLCB resistance gene, conferring partial resistance, is currently known, underscoring the importance of isolating new SCLB resistance-related genes. Here, we performed a comparative proteomic analysis and identified 258 proteins showing differential abundance during the maize response to B. maydis. These proteins included an ascorbate peroxidase (Zea mays ascorbate peroxidase 1 (ZmAPX1)) encoded by a gene located within the mapping interval of a previously identified quantitative trait locus associated with SCLB resistance. ZmAPX1 overexpression resulted in lower H2O2 accumulation and enhanced resistance against B. maydis. Jasmonic acid (JA) contents and transcript levels for JA biosynthesis and responsive genes increased in ZmAPX1-overexpressing plants infected with B. maydis, whereas Zmapx1 mutants showed the opposite effects. We further determined that low levels of H2O2 are accompanied by an accumulation of JA that enhances SCLB resistance. These results demonstrate that ZmAPX1 positively regulates SCLB resistance by decreasing H2O2 accumulation and activating the JA-mediated defense signaling pathway. This study identified ZmAPX1 as a potentially useful gene for increasing SCLB resistance. Furthermore, the generated data may be relevant for clarifying the functions of plant APXs.
Sorghum, the fifth largest cereal crop, has high value as a staple food and raw material for liquor and vinegar brewing. Due to its high biomass and quality, it is also used as the second most planted silage resource. No fragrant sorghums are currently on the market. Through CRISPR/Cas9-mediated knockout of SbBADH2, we obtained sorghum lines with extraordinary aromatic smell in both seeds and leaves. Animal feeding experiments showed that fragrant sorghum leaves were attractable. We believe this advantage will produce great value in the sorghum market for both grain and whole biomass forage.
The transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5) plays a crucial role in abscisic acid (ABA) signaling during seed germination. However, how ABI5 is regulated during this process is poorly understood. Here, we report that the ubiquitin E3 ligase MIEL1 and its target transcription factor MYB30 modulate ABA responses in Arabidopsis thaliana during seed germination and seedling establishment via the precise regulation of ABI5. MIEL1 interacts with and ubiquitinates ABI5 to facilitate its degradation during germination. The transcription factor MYB30, whose turnover is mediated by MIEL1 during seed germination, also interacts with ABI5 to interfere with its transcriptional activity. MYB30 functions downstream of MIEL1 in the ABA response, and both are epistatic to ABI5 in ABA-mediated inhibition of seed germination and postgerminative growth. ABA treatment induces the degradation of MIEL1 and represses the interaction between MIEL1 and ABI5/MYB30, thus releasing both ABI5 and MYB30. Our results demonstrate that MIEL1 directly mediates the proteasomal degradation of ABI5 and inhibits its activity via the release of its target protein MYB30, thus ensuring precise ABA signaling during seed germination and seedling establishment.
The mechanisms that balance plant growth and stress responses are poorly understood, but they appear to involve abscisic acid (ABA) signaling mediated by protein kinases. Here, to explore these mechanisms, we examined the responses of Arabidopsis thaliana protein kinase mutants to ABA treatment. We found that mutants of BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) were hypersensitive to the effects of ABA on both seed germination and primary root growth. The kinase OPEN STOMATA 1 (OST1) was more highly activated by ABA in bak1 mutant than the wild type. BAK1 was not activated by ABA treatment in the dominant negative mutant abi1-1 or the pyr1 pyl4 pyl5 pyl8 quadruple mutant, but it was more highly activated by this treatment in the abi1-2 abi2-2 hab1-1 loss-of-function triple mutant than the wild type. BAK1 phosphorylates OST1 T146 and inhibits its activity. Genetic analyses suggested that BAK1 acts at or upstream of core components in the ABA signaling pathway, including PYLs, PP2Cs, and SnRK2s, during seed germination and primary root growth. Although the upstream brassinosteroid (BR) signaling components BAK1 and BR INSENSITIVE 1 (BRI1) positively regulate ABA-induced stomatal closure, mutations affecting downstream components of BR signaling, including BRASSINOSTEROID-SIGNALING KINASEs (BSKs) and BRASSINOSTEROID-INSENSITIVE 2 (BIN2), did not affect ABA-mediated stomatal movement. Thus, our study uncovered an important role of BAK1 in negatively regulating ABA signaling during seed germination and primary root growth, but positively modulating ABA-induced stomatal closure, thus optimizing the plant growth under drought stress.
Anther development from stamen primordium to pollen dispersal is complex and essential to sexual reproduction. How this highly dynamic and complex developmental process is controlled genetically is not well understood, especially for genes involved in specific key developmental phases. Here we generated RNA sequencing libraries spanning 10 key stages across the entirety of anther development in maize (Zea mays). Global transcriptome analyses revealed distinct phases of cell division and expansion, meiosis, pollen maturation, and mature pollen, for which we detected 50, 245, 42, and 414 phase-specific marker genes, respectively. Phase-specific transcription factor genes were significantly enriched in the phase of meiosis. The phase-specific expression of these marker genes was highly conserved among the maize lines Chang7-2 and W23, indicating they might have important roles in anther development. We explored a desiccation-related protein gene, ZmDRP1, which was exclusively expressed in the tapetum from the tetrad to the uninucleate microspore stage, by generating knockout mutants. Notably, mutants in ZmDRP1 were completely male-sterile, with abnormal Ubisch bodies and defective pollen exine. Our work provides a glimpse into the gene expression dynamics and a valuable resource for exploring the roles of key phase-specific genes that regulate anther development.
Iron (Fe) homeostasis is critical for plant growth, development, and stress responses. Fe levels are tightly controlled by intricate regulatory networks in which transcription factors (TFs) play a central role. A series of basic helix-loop-helix (bHLH) TFs have been shown to contribute to Fe homeostasis, but the regulatory layers beyond bHLH TFs remain largely unclear. Here, we demonstrate that the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) TF SlSPL-CNR negatively regulates Fe-deficiency responses in tomato (Solanum lycopersicum) roots. Fe deficiency rapidly repressed the expression of SlSPL-CNR, and Fe deficiency responses were intensified in two clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-generated SlSPL-CNR knock-out lines compared to the wild-type. Comparative transcriptome analysis identified 47 Fe deficiency-responsive genes the expression of which is negatively regulated by SlSPL-CNR, one of which, SlbHLH101, helps regulate Fe uptake genes. SlSPL-CNR localizes the nucleus and interacts with the GTAC and BOX 4 (ATTAAT) motifs in the SlbHLH101 promoter to repress its expression. Inhibition of SlSPL-CNR expression in response to Fe deficiency was well correlated with the expression of the microRNA SlymiR157. SlymiR157-overexpressing tomato lines displayed enhanced Fe deficiency responses, as did SlSPL-CNR loss-of-function mutants. We propose that the SlymiR157-SlSPL-CNR module represents a novel pathway that acts upstream of SlbHLH101 to regulate Fe homeostasis in tomato roots.
Heterosis is a fundamental biological phenomenon characterized by the superior performance of hybrids over their parents. Although tremendous progress has been reported in seed crops, the molecular mechanisms underlying heterosis in clonally propagated crops are largely unknown. Potato (Solanum tuberosum L.) is the most important tuber crop and an ongoing revolution is transforming potato from a clonally propagated tetraploid crop into a seed-propagated diploid hybrid potato. In our previous study, we developed the first generation of highly homozygous inbred lines of potato and hybrids with strong heterosis. Here, we integrated transcriptome, metabolome, and DNA methylation data to explore the genetic and molecular basis of potato heterosis at three developmental stages. We found that the initial establishment of heterosis in diploid potato was mainly due to dominant complementation. Flower color, male fertility, and starch and sucrose metabolism showed obvious gene dominant complementation in hybrids, and hybrids devoted more energy to primary metabolism for rapid growth. In addition, we identified ~2 700 allele-specific expression genes at each stage, which likely function in potato heterosis and might be regulated by CHH allele-specific methylation level. Our multi-omics analysis provides insight into heterosis in potato and facilitates the exploitation of heterosis in potato breeding.
Here, through single-molecule real-time sequencing, we present a high-quality genome sequence of the Japanese larch (Larix kaempferi), a conifer species with great value for wood production and ecological afforestation. The assembled genome is 10.97 Gb in size, harboring 45,828 protein-coding genes. Of the genome, 66.8% consists of repeat sequences, of which long terminal repeat retrotransposons are dominant and make up 69.86%. We find that tandem duplications have been responsible for the expansion of genes involved in transcriptional regulation and stress responses, unveiling their crucial roles in adaptive evolution. Population transcriptome analysis reveals that lignin content in L. kaempferi is mainly determined by the process of monolignol polymerization. The expression values of six genes (LkCOMT7, LkCOMT8, LkLAC23, LkLAC102, LkPRX148, and LkPRX166) have significantly positive correlations with lignin content. These results indicated that the increased expression of these six genes might be responsible for the high lignin content of the larches' wood. Overall, this study provides new genome resources for investigating the evolution and biological function of conifer trees, and also offers new insights into wood properties of larches.
Glycogen synthase kinase 3 (GSK3) proteins play key roles in brassinosteroid (BR) signaling during plant growth and development by phosphorylating various substrates. However, how GSK3 protein stability and activity are themselves modulated is not well understood. Here, we demonstrate in vitro and in vivo that C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 (OsCPL3), a member of the RNA Pol II CTD phosphatase-like family, physically interacts with OsGSK2 in rice (Oryza sativa). OsCPL3 expression was widely detected in various tissues and organs including roots, leaves and lamina joints, and was induced by exogenous BR treatment. OsCPL3 localized to the nucleus, where it dephosphorylated OsGSK2 at the Ser-222 and Thr-284 residues to modulate its protein turnover and kinase activity, in turn affecting the degradation of BRASSINAZOLE-RESISTANT 1 (BZR1) and BR signaling. Loss of OsCPL3 function resulted in higher OsGSK2 abundance and lower OsBZR1 levels, leading to decreased BR responsiveness and alterations in plant morphology including semi-dwarfism, leaf erectness and grain size, which are of fundamental importance to crop productivity. These results reveal a previously unrecognized role for OsCPL3 and add another layer of complexity to the tightly controlled BR signaling pathway in plants.
The development of plants is largely dependent on their growth environment. To better adapt to a particular habitat, plants have evolved various subtle regulatory mechanisms for altering gene expression. Non coding RNAs (ncRNAs) constitute a major portion of the transcriptomes of eukaryotes. Various ncRNAs have been recognized as important regulators of the expression of genes involved in essential biological processes throughout the whole life cycles of plants. In this review, we summarize the current understanding of the biogenesis and contributions of small nucle olar RNA (snoRNA)- and regulatory long non coding RNA (lncRNA)-mediated gene regulation in plant development and environmental responses. Many regulatory ncRNAs appear to be associated with increased yield, quality and disease resistance of various species and cultivars. These ncRNAs may potentially be used as genetic resources for improving agronomic traits and for molecular breeding. The challenges in understanding plant ncRNA biology and the possibilities to make better use of these valuable gene resources in the future are discussed in this review.
Jasmonic acid (JA) is a key regulator of plant defense responses. Although the transcription factor MYC2, the master regulator of the JA signaling pathway, orchestrates a hierarchical transcriptional cascade that regulates the JA responses, only a few transcriptional regulators involved in this cascade have been described. Here, we identified the basic helix-loop-helix (bHLH) transcription factor gene in tomato (Solanum lycopersicum), METHYL JASMONATE (MeJA)-INDUCED GENE (SlJIG), the expression of which was strongly induced by MeJA treatment. Genetic and molecular biology experiments revealed that SlJIG is a direct target of MYC2. SlJIG knockout plants generated by gene editing had lower terpene contents than the wild type from the lower expression of TERPENE SYNTHASE (TPS) genes, rendering them more appealing to cotton bollworm (Helicoverpa armigera). Moreover, SlJIG knockouts exhibited weaker JA-mediated induction of TPSs, suggesting that SlJIG may participate in JA-induced terpene biosynthesis. Knocking out SlJIG also resulted in attenuated expression of JA-responsive defense genes, which may contribute to the observed lower resistance to cotton bollworm and to the fungus Botrytis cinerea. We conclude that SlJIG is a direct target of MYC2, forms a MYC2-SlJIG module, and functions in terpene biosynthesis and resistance against cotton bollworm and B. cinerea.
For Upcoming Special lssue: