Comparative study of two indoor microbial volatile pollutants, 2-Methyl-1-butanol and 3-Methyl-1-butanol, on growth and antioxidant system of rice (Oryza sativa) seedlings.

2-Methyl-1-butanol (2MB) and 3-Methyl-1-butanol (3MB) are microbial volatile organic compounds (VOCs) and found in indoor air. Here, we applied rice as a bioindicator to investigate the effects of these indoor microbial volatile pollutants. A remarkable decrease in germination percentage, shoot and root elongation, as well as lateral root numbers were observed in 3MB. Furthermore, ROS production increased by 2MB and 3MB, suggesting that pentanol isomers could induce cytotoxicity in rice seedlings. The enhancement of peroxidase (POD) and catalase (CAT) activity provided evidence that pentanol isomers activated the enzymatic antioxidant scavenging systems, with a more significant effect observed in 3MB. Furthermore, 3MB induced higher activity levels of glutathione (GSH), oxidized glutathione (GSSG), and the GSH/GSSG ratio in rice compared to the levels induced by 2MB. Additionally, qRT-PCR analysis showed more up-regulation in the expression of glutaredoxins (GRXs), peroxiredoxins (PRXs), thioredoxins (TRXs), and glutathione S-transferases (GSTUs) genes in 3MB. Taking the impacts of pentanol isomers together, the present study suggests that 3MB exhibits more cytotoxic than 2MB, as such has critical effects on germination and the early seedling stage of rice. Our results provide molecular insights into how isomeric indoor microbial volatile pollutants affect plant growth through airborne signals.

Signaling pathways involved in microbial indoor air pollutant 3-methyl-1-butanol in the induction of stomatal closure in Arabidopsis.

Indoor air pollution is a global problem and one of the main stress factors that has negative effects on plant and human health. 3-methyl-1-butanol (3MB), an indoor air pollutant, is a microbial volatile organic compound (mVOC) commonly found in damp indoor dwellings. In this study, we reported that 1 mg/L of 3MB can elicit a significant reduction in the stomatal aperture ratio in Arabidopsis and tobacco. Our results also showed that 3MB enhances the reactive oxygen species (ROS) production in guard cells of wild-type Arabidopsis after 24 h exposure. Further investigation of 24 h 3MB fumigation of rbohD, the1-1, mkk1, mkk3, and nced3 mutants revealed that ROS production, cell wall integrity, MAPK kinases cascade, and phytohormone abscisic acid are all involved in the process of 3MB-induced stomatal. Our findings proposed a mechanism by which 3MB regulates stomatal closure in Arabidopsis. Understanding the mechanisms by which microbial indoor air pollutant induces stomatal closure is critical for modulating the intake of harmful gases from indoor environments into leaves. Investigations into how stomata respond to the indoor mVOC 3MB will shed light on the plant's "self-defense" system responding to indoor air pollution.

Arabidopsis transcription factor TCP13 promotes shade avoidance syndrome-like responses by directly targeting a subset of shade-responsive gene promoters.

TCP13 belongs to a subgroup of TCP transcription factors implicated in the shade avoidance syndrome (SAS), but its exact role remains unclear. Here, we show that TCP13 promotes the SAS-like response by enhancing hypocotyl elongation and suppressing flavonoid biosynthesis as a part of the incoherent feed-forward loop in light signaling. Shade is known to promote the SAS by activating PHYTOCHROME-INTERACTING FACTOR (PIF)-auxin signaling in plants, but we found no evidence in a transcriptome analysis that TCP13 activates PIF-auxin signaling. Instead, TCP13 mimics shade by activating the expression of a subset of shade-inducible and cell elongation-promoting SAUR genes including SAUR19, by direct targeting of their promoters. We also found that TCP13 and PIF4, a molecular proxy for shade, repress the expression of flavonoid biosynthetic genes by directly targeting both shared and distinct sets of biosynthetic gene promoters. Together, our results indicate that TCP13 promotes the SAS-like response by directly targeting a subset of shade-responsive genes without activating the PIF-auxin signaling pathway.

Antifungal mechanism of volatile compounds emitted by Actinomycetota Paenarthrobacter ureafaciens from a disease-suppressive soil on Saccharomyces cerevisiae.

Increasing evidence suggests that in disease-suppressive soils, microbial volatile compounds (mVCs) released from bacteria may inhibit the growth of plant-pathogenic fungi. However, the antifungal activities and molecular responses of fungi to different mVCs remain largely undescribed. In this study, we first evaluated the responses of pathogenic fungi to treatment with mVCs from Paenarthrobacter ureafaciens. Then, we utilized the well-characterized fungal model organism Saccharomyces cerevisiae to study the potential mechanistic effects of the mVCs. Our data showed that exposure to P. ureafaciens mVCs leads to reduced growth of several pathogenic fungi, and in yeast cells, mVC exposure prompts the accumulation of reactive oxygen species. Further experiments with S. cerevisiae deletion mutants indicated that Slt2/Mpk1 and Hog1 MAPKs play major roles in the yeast response to P. ureafaciens mVCs. Transcriptomic analysis revealed that exposure to mVCs was associated with 1,030 differentially expressed genes (DEGs) in yeast. According to gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses, many of these DEGs are involved in mitochondrial dysfunction, cell integrity, mitophagy, cellular metabolism, and iron uptake. Genes encoding antimicrobial proteins were also significantly altered in the yeast after exposure to mVCs. These findings suggest that oxidative damage and mitochondrial dysfunction are major contributors to the fungal toxicity of mVCs. Furthermore, our data showed that cell wall, antioxidant, and antimicrobial defenses are induced in yeast exposed to mVCs. Thus, our findings expand upon previous research by delineating the transcriptional responses of the fungal model. IMPORTANCE Since the use of bacteria-emitted volatile compounds in phytopathogen control is of considerable interest, it is important to understand the molecular mechanisms by which fungi may adapt to microbial volatile compounds (mVCs). Paenarthrobacter ureafaciens is an isolated bacterium from disease-suppressive soil that belongs to the Actinomycetota phylum. P. ureafaciens mVCs showed a potent antifungal effect on phytopathogens, which may contribute to disease suppression in soil. However, our knowledge about the antifungal mechanism of mVCs is limited. This study has proven that mVCs are toxic to fungi due to oxidative stress and mitochondrial dysfunction. To deal with mVC toxicity, antioxidants and physical defenses are required. Furthermore, iron uptake and CAP proteins are required for antimicrobial defense, which is necessary for fungi to deal with the thread from mVCs. This study provides essential foundational knowledge regarding the molecular responses of fungi to inhibitory mVCs.

Transcriptome analyses uncover reliance of endosperm gene expression on Arabidopsis embryonic development.

Endosperm-embryo development in flowering plants is regulated coordinately by signal exchange during seed development. However, such a reciprocal control mechanism has not been clearly identified. In this study, we identified an endosperm-specific gene, LBD35, expressed in an embryonic development-dependent manner, by a comparative transcriptome and cytological analyses of double-fertilized and single-fertilized seeds prepared by using the kokopelli mutant, which frequently induces single fertilization events. Transcriptome analysis using LBD35 as a marker of the central cell fertilization event identified that 141 genes, including 31 genes for small cysteine-rich peptides, are expressed in a double fertilization-dependent manner. Our results reveal possible embryonic signals that regulate endosperm gene expression and provide a practicable method to identify genes involved in the communication during endosperm-embryo development.

Transcription factors KNAT3 and KNAT4 are essential for integument and ovule formation in Arabidopsis.

Integuments form important protective cell layers surrounding the developing ovules in gymno- and angiosperms. Although several genes have been shown to influence the development of integuments, the transcriptional regulatory mechanism is still poorly understood. In this work, we report that the Class II KNOTTED1-LIKE HOMEOBOX (KNOX II) transcription factors KNOTTED1-LIKE HOMEBOX GENE 3 (KNAT3) and KNAT4 regulate integument development in Arabidopsis (Arabidopsis thaliana). KNAT3 and KNAT4 were co-expressed in inflorescences and especially in young developing ovules. The loss-of-function double mutant knat3 knat4 showed an infertility phenotype, in which both inner and outer integuments of the ovule are arrested at an early stage and form an amorphous structure as in the bell1 (bel1) mutant. The expression of chimeric KNAT3- and KNAT4-EAR motif repression domain (SRDX repressors) resulted in severe seed abortion. Protein-protein interaction assays demonstrated that KNAT3 and KNAT4 interact with each other and also with INNER NO OUTER (INO), a key transcription factor required for the outer integument formation. Transcriptome analysis showed that the expression of genes related with integument development is influenced in the knat3 knat4 mutant. The knat3 knat4 mutant also had a lower indole-3-acetic acid (IAA) content, and some auxin signaling pathway genes were downregulated. Moreover, transactivation analysis indicated that KNAT3/4 and INO activate the auxin signaling gene IAA INDUCIBLE 14 (IAA14). Taken together, our study identified KNAT3 and KNAT4 as key factors in integument development in Arabidopsis.

Elongation of Siliques Without Pollination 3 Regulates Nutrient Flow Necessary for Embryogenesis.

Apomixis, defined as the transfer of maternal germplasm to offspring without fertilization, enables the fixation of F1-useful traits, providing advantages in crop breeding. However, most apomictic plants require pollination to produce the endosperm. The endosperm is essential for embryogenesis, and its development is suppressed until fertilization. We show that the expression of a chimeric repressor of the Elongation of Siliques without Pollination 3 (ESP3) gene (Pro35S:ESP3-SRDX) induces ovule enlargement without fertilization in Arabidopsis thaliana. The ESP3 gene encodes a protein similar to the flowering Wageningen homeodomain transcription factor containing a StAR-related lipid transfer domain. However, ESP3 lacks the homeobox-encoding region. Genes related to the cell cycle and sugar metabolism were upregulated in unfertilized Pro35S:ESP3-SRDX ovules similar to those in fertilized seeds, while those related to autophagy were downregulated similar to those in fertilized seeds. Unfertilized Pro35S:ESP3-SRDX ovules partially nourished embryos when only the egg was fertilized, accumulating hexoses without central cell proliferation. ESP3 may regulate nutrient flow during seed development, and ESP3-SRDX could be a useful tool for complete apomixis that does not require pseudo-fertilization.

The involvement of AtMKK1 and AtMKK3 in plant-deleterious microbial volatile compounds-induced defense responses.

KEY MESSAGE: Plant-deleterious microbial volatiles activate the transactivation of hypoxia, MAMPs and wound responsive genes in Arabidopsis thaliana. AtMKK1 and AtMKK3 are involved in the plant-deleterious microbial volatiles-induced defense responses. Microbial volatile compounds (mVCs) are a collection of volatile metabolites from microorganisms with biological effects on all living organisms. mVCs function as gaseous modulators of plant growth and plant health. In this study, the defense events induced by plant-deleterious mVCs were investigated. Enterobacter aerogenes VCs lead to growth inhibition and immune responses in Arabidopsis thaliana. E. aerogenes VCs negatively regulate auxin response and transport gene expression in the root tip, as evidenced by decreased expression of DR5::GFP, PIN3::PIN3-GFP and PIN4::PIN4-GFP. Data from transcriptional analysis suggests that E. aerogenes VCs trigger hypoxia response, innate immune responses and metabolic processes. In addition, the transcript levels of the genes involved in the synthetic pathways of antimicrobial metabolites camalexin and coumarin are increased after the E. aerogenes VCs exposure. Moreover, we demonstrate that MKK1 serves as a regulator of camalexin biosynthesis gene expression in response to E. aerogenes VCs, while MKK3 is the regulator of coumarin biosynthesis gene expression. Additionally, MKK1 and MKK3 mediate the E. aerogenes VCs-induced callose deposition. Collectively, these studies provide molecular insights into immune responses by plant-deleterious mVCs.

Optimal culture conditions of Piriformospora indica for volatile compound release to promote effective plant growth.

Piriformospora indica, which is an endophytic fungus that grows on various media in the absence of a host, emits plant growth promoting volatile organic compounds (VOCs). Kaefer medium (KF) has been shown to be the most suitable medium for P. indica growth; however, different media may differentially affect fungal metabolism which may in turn influence the VOC profiles of P. indica. To date, how the VOCs emitted from P. indica cultured on different media affect plant growth has not been well characterized. Here, we show that poor nutrient medium (PNM) promoted the growth of P. indica more effectively than potato dextrose agar (PDA) or KF medium. By contrast, plant total biomass and root fresh weight were increased 1.8-fold and 2.1-fold, when co-cultivated with P. indica cultured on PDA medium in comparison with KF or PNM medium, respectively. Furthermore, sucrose in the plant culture medium downregulated the fold-induction ratio of the plant growth promoted by P. indica VOCs.

Arabidopsis zinc finger homeodomain transcription factor BRASSINOSTEROID-RELATED HOMEOBOX 2 acts as a positive regulator of brassinosteroid response.

The brassinosteroid (BR) phytohormone is an important regulator of plant growth. To identify novel transcription factors that regulate BR responses, we screened chimeric repressor gene silencing technology (CRES-T) plants, in which transcription factors were converted into chimeric repressors by the fusion of SRDX plant-specific repression domain, with brassinazole (Brz), an inhibitor of BR biosynthesis. We identified that a line that expressed the chimeric repressor for zinc finger homeobox transcription factor, BRASSINOSTEORID-RELATED-HOMEOBOX-2 (BHB2-sx), exhibited Brz-hypersensitive phenotype with shorter hypocotyl under dark, dwarf and round and dark green leaves similar to BR-deficient phenotype. Similar to BHB2-sx plants, bhb2 knockout mutant also exhibited Brz hypersensitive phenotype. In contrast, ectopic expression of BHB2 (BHB2-ox) showed hypocotyl elongation phenotype (BR excessive), showing decrease to Brz sensitivity. The expression of the DWF4 and CPD BR biosynthesis genes was repressed in BHB2-sx plants, whereas it was enhanced in BHB2-ox plants. The BR deficient-like phenotype of BHB2-sx plants was partially restored by treatment with brassinolide (BL), indicating that the BR deficient phenotype of BHB2-sx plant may be due to suppression of BR biosynthesis. Our results indicate that BHB2 is a positive regulator of BR response may be due to the promotion of BR biosynthesis genes.

Arabidopsis homeobox-leucine zipper transcription factor BRASSINOSTEROID-RELATED HOMEOBOX 3 regulates leaf greenness by suppressing BR signaling.

Brassinosteroid (BR) is a phytohormone that acts as important regulator of plant growth. To identify novel transcription factors that may be involved in unknown mechanisms of BR signaling, we screened the chimeric repressor expressing plants (CRES-T), in which transcription factors were converted into chimeric repressors by the fusion of SRDX plant-specific repression domain, to identify those that affect the expression of BR inducible genes. Here, we identified a homeobox-leucine zipper type transcription factor, BRASSINOSTEROID-RELATED-HOMEOBOX 3 (BHB3), of which a chimeric repressor expressing plants (BHB3-sx) significantly downregulated the expression of BAS1 and SAUR-AC1 that are BR inducible genes. Interestingly, ectopic expression of BHB3 (BHB3-ox) also repressed the BR inducible genes and shorten hypocotyl that would be similar to a BR-deficient phenotype. Interestingly, both BHB3-sx and BHB3-ox showed pale green phenotype, in which the expression of genes related photosynthesis and chlorophyll contents were significantly decreased. We found that BHB3 contains three motifs similar to the conserved EAR-repression domain, suggesting that BHB3 may act as a transcriptional repressor. These results indicate that BHB3 might play an important role not only to the BR signaling but also the regulation of greenings.

A collection of inducible transcription factor-glucocorticoid receptor fusion lines for functional analyses in Arabidopsis thaliana.

Arabidopsis possesses approximately 2000 transcription factors (TFs) in its genome. They play pivotal roles in various biological processes but analysis of their function has been hampered by the overlapping nature of their activities. To uncover clues to their function, we generated inducible TF lines using glucocorticoid receptor (GR) fusion techniques in Arabidopsis. These TF-GR lines each express one of 1255 TFs as a fusion with the GR gene. An average 14 lines of T2 transgenic TF-GR lines were generated for each TF to monitor their function. To evaluate these transcription lines, we induced the TF-GR lines of phytochrome-interacting factor 4, which controls photomorphogenesis, with synthetic glucocorticoid dexamethasone. These phytochrome-interacting factor 4-GR lines showed the phenotype described in a previous report. We performed screening of the other TF-GR lines for TFs involved in light signaling under blue and far-red light conditions and identified 13 novel TF candidates. Among these, we found two lines showing higher anthocyanin accumulation under light conditions and we examined the regulating genes. These results indicate that the TF-GR lines can be used to dissect functionally redundant genes in plants and demonstrate that the TF-GR line collection can be used as an effective tool for functional analysis of TFs.

Low nitrogen conditions accelerate flowering by modulating the phosphorylation state of FLOWERING BHLH 4 in Arabidopsis.

Nitrogen (N) is an essential nutrient that affects multiple plant developmental processes, including flowering. As flowering requires resources to develop sink tissues for reproduction, nutrient availability is tightly linked to this process. Low N levels accelerate floral transition; however, the molecular mechanisms underlying this response are not well understood. Here, we identify the FLOWERING BHLH 4 (FBH4) transcription factor as a key regulator of N-responsive flowering in Arabidopsis Low N-induced early flowering is compromised in fbh quadruple mutants. We found that FBH4 is a highly phosphorylated protein and that FBH4 phosphorylation levels decrease under low N conditions. In addition, decreased phosphorylation promotes FBH4 nuclear localization and transcriptional activation of the direct target CONSTANS (CO) and downstream florigen FLOWERING LOCUS T (FT) genes. Moreover, we demonstrate that the evolutionarily conserved cellular fuel sensor SNF1-RELATED KINASE 1 (SnRK1), whose kinase activity is down-regulated under low N conditions, directly phosphorylates FBH4. SnRK1 negatively regulates CO and FT transcript levels under high N conditions. Together, these results reveal a mechanism by which N levels may fine-tune FBH4 nuclear localization by adjusting the phosphorylation state to modulate flowering time. In addition to its role in flowering regulation, we also showed that FBH4 was involved in low N-induced up-regulation of nutrient recycling and remobilization-related gene expression. Thus, our findings provide insight into N-responsive growth phase transitions and optimization of plant fitness under nutrient-limited conditions.

Two types of bHLH transcription factor determine the competence of the pericycle for lateral root initiation.

The molecular basis of the competence of the pericycle cell to initiate lateral root primordium formation is totally unknown. Here, we report that in Arabidopsis, two types of basic helix-loop-helix (bHLH) transcription factors, named PERICYCLE FACTOR TYPE-A (PFA) proteins and PERICYCLE FACTOR TYPE-B (PFB) proteins, govern the competence of pericycle cells to initiate lateral root primordium formation. Overexpression of PFA genes confers hallmark pericycle characteristics, including specific marker gene expression and auxin-induced cell division, and multiple loss-of-function mutations in PFA genes or the repression of PFB target genes results in the loss of this specific pericycle function. PFA and PFB proteins physically interact and are under mutual- and self-regulation, forming a positive feedback loop. This study unveils the transcriptional regulatory system that determines pericycle participation in lateral root initiation.

Mutation of the imprinted gene OsEMF2a induces autonomous endosperm development and delayed cellularization in rice.

In angiosperms, endosperm development comprises a series of developmental transitions controlled by genetic and epigenetic mechanisms that are initiated after double fertilization. Polycomb repressive complex 2 (PRC2) is a key component of these mechanisms that mediate histone H3 lysine 27 trimethylation (H3K27me3); the action of PRC2 is well described in Arabidopsis thaliana but remains uncertain in cereals. In this study, we demonstrate that mutation of the rice (Oryza sativa) gene EMBRYONIC FLOWER2a (OsEMF2a), encoding a zinc-finger containing component of PRC2, causes an autonomous endosperm phenotype involving proliferation of the central cell nuclei with separate cytoplasmic domains, even in the absence of fertilization. Detailed cytological and transcriptomic analyses revealed that the autonomous endosperm can produce storage compounds, starch granules, and protein bodies specific to the endosperm. These events have not been reported in Arabidopsis. After fertilization, we observed an abnormally delayed developmental transition in the endosperm. Transcriptome and H3K27me3 ChIP-seq analyses using endosperm from the emf2a mutant identified downstream targets of PRC2. These included >100 transcription factor genes such as type-I MADS-box genes, which are likely required for endosperm development. Our results demonstrate that OsEMF2a-containing PRC2 controls endosperm developmental programs before and after fertilization.

Cellular dynamics of double fertilization and early embryogenesis in flowering plants.

Flowering plants (angiosperms) perform a unique double fertilization in which two sperm cells fuse with two female gamete cells in the embryo sac to develop a seed. Furthermore, during land plant evolution, the mode of sexual reproduction has been modified dramatically from motile sperm in the early-diverging land plants, such as mosses and ferns as well as some gymnosperms (Ginkgo and cycads) to nonmotile sperm that are delivered to female gametes by the pollen tube in flowering plants. Recent studies have revealed the cellular dynamics and molecular mechanisms for the complex series of double fertilization processes and elucidated differences and similarities between animals and plants. Here, together with a brief comparison with animals, we review the current understanding of flowering plant zygote dynamics, covering from gamete nuclear migration, karyogamy, and polyspermy block, to zygotic genome activation as well as asymmetrical division of the zygote. Further analyses of the detailed molecular and cellular mechanisms of flowering plant fertilization should shed light on the evolution of the unique sexual reproduction of flowering plants.

The CIB1 transcription factor regulates light- and heat-inducible cell elongation via a two-step HLH/bHLH system.

Light and high temperature promote plant cell elongation. PHYTOCHROME INTERACTING FACTOR4 (PIF4, a typical basic helix-loop-helix [bHLH] transcriptional activator) and the non-DNA binding atypical HLH inhibitors PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and LONG HYPOCOTYL IN FAR-RED 1 (HFR1) competitively regulate cell elongation in response to light conditions and high temperature. However, the underlying mechanisms have not been fully clarified. Here, we show that in Arabidopsis thaliana, the bHLH transcription factor CRYPTOCHROME-INTERACTING BASIC HELIX-LOOP-HELIX 1 (CIB1) positively regulates cell elongation under the control of PIF4, PAR1, and HFR1. Furthermore, PIF4 directly regulates CIB1 expression by interacting with its promoter, and PAR1 and HFR1 interfere with PIF4 binding to the CIB1 promoter. CIB1 activates genes that function in cell elongation, and PAR1 interferes with the DNA binding activity of CIB1, thus suppressing cell elongation. Hence, two antagonistic HLH/bHLH systems, the PIF4-PAR1/HFR1 and CIB1-PAR1 systems, regulate cell elongation in response to light and high temperature. We thus demonstrate the important role of non-DNA binding small HLH proteins in the transcriptional regulation of cell elongation in plants.

Improving the Efficiency of Adventitious Shoot Induction and Somatic Embryogenesis via Modification of WUSCHEL and LEAFY COTYLEDON 1.

The induction of adventitious organs, such as calli, shoots, and somatic embryos, in tissue culture is a useful technique for plant propagation and genetic modification. In recent years, several genes have been reported to be adventitious organ inducers and proposed to be useful for industrial applications. Even though the Arabidopsis (Arabidopsis thaliana) WUSCHEL (WUS) and LEAFY COTYLEDON 1 (LEC1) genes can induce adventitious organ formation in Arabidopsis without phytohormone treatment, further improvement is desired. Here, we show that modifying the transcriptional repression/activation activities of WUS and LEC1 improves the efficiency of adventitious organ formation in Arabidopsis. Because WUS functions as a transcriptional repressor during the induction of adventitious organs, we fused it to an artificial strong repression domain, SUPERMAN REPRESSION DOMAIN X (SRDX). Conversely, we fused the strong transcriptional activation domain VP16 from herpes simplex virus to LEC1. Upon overexpression of the corresponding transgenes, we succeeded in improving the efficiency of adventitious organ induction. Our results show that the modification of transcriptional repression/activation activity offers an effective method to improve the efficiency of adventitious organ formation in plants.

Author Correction: The Apostasia genome and the evolution of orchids.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The class II KNOX transcription factors KNAT3 and KNAT7 synergistically regulate monolignol biosynthesis in Arabidopsis.

The function of the transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is still unclear since it appears to be either a negative or a positive regulator for secondary cell wall deposition with its loss-of-function mutant displaying thicker interfascicular and xylary fiber cell walls but thinner vessel cell walls in inflorescence stems. To explore the exact function of KNAT7, class II KNOTTED1-LIKE HOMEOBOX (KNOX II) genes in Arabidopsis including KNAT3, KNAT4, and KNAT5 were studied together. By chimeric repressor technology, we found that both KNAT3 and KNAT7 repressors exhibited a similar dwarf phenotype. Both KNAT3 and KNAT7 genes were expressed in the inflorescence stems and the knat3 knat7 double mutant exhibited a dwarf phenotype similar to the repressor lines. A stem cross-section of knat3 knat7 displayed an enhanced irregular xylem phenotype as compared with the single mutants, and its cell wall thickness in xylem vessels and interfascicular fibers was significantly reduced. Analysis of cell wall chemical composition revealed that syringyl lignin was significantly decreased while guaiacyl lignin was increased in the knat3 knat7 double mutant. Coincidently, the knat3 knat7 transcriptome showed that most lignin pathway genes were activated, whereas the syringyl lignin-related gene Ferulate 5-Hydroxylase (F5H) was down-regulated. Protein interaction analysis revealed that KNAT3 and KNAT7 can form a heterodimer, and KNAT3, but not KNAT7, can interact with the key secondary cell wall formation transcription factors NST1/2, which suggests that the KNAT3-NST1/2 heterodimer complex regulates F5H to promote syringyl lignin synthesis. These results indicate that KNAT3 and KNAT7 synergistically work together to promote secondary cell wall biosynthesis.