Brassinosteroids (BRs) are phytosteroid hormones controlling various physiological processes critical for normal growth and development. BRs are perceived by a protein complex containing two transmembrane receptor kinases, BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) [1-3]. BRI1 null mutants exhibit a dwarfed stature with epinastic leaves, delayed senescence, reduced male fertility, and altered light responses. BAK1 null mutants, however, only show a subtle phenotype, suggesting that functionally redundant proteins might be present in the Arabidopsis genome. Here we report that BAK1-LIKE 1 (BKK1) functions redundantly with BAK1 in regulating BR signaling. Surprisingly, rather than the expected bri1-like phenotype, bak1 bkk1 double mutants exhibit a seedling-lethality phenotype due to constitutive defense-gene expression, callose deposition, reactive oxygen species (ROS) accumulation, and spontaneous cell death even under sterile growing conditions. Our detailed analyses demonstrate that BAK1 and BKK1 have dual physiological roles: positively regulating a BR-dependent plant growth pathway, and negatively regulating a BR-independent cell-death pathway. Both BR signaling and developmentally controlled cell death are critical to optimal plant growth and development, but the mechanisms regulating early events in these pathways are poorly understood. This study provides novel insights into the initiation and crosstalk of the two signaling cascades.
The Arabidopsis thaliana Somatic Embryogenesis Receptor Kinases (SERKs) consist of five members, SERK1 to SERK5, of the leucine-rich repeat receptor-like kinase subfamily II (LRR-RLK II). SERK3 was named BRI1-Associated Receptor Kinase 1 (BAK1) due to its direct interaction with the brassinosteroid (BR) receptor BRI1 in vivo, while SERK4 has also been designated as BAK1-Like 1 (BKK1) for its functionally redundant role with BAK1. Here we provide genetic and biochemical evidence to demonstrate that SERKs are absolutely required for early steps in BR signaling. Overexpression of four of the five SERKs—SERK1, SERK2, SERK3/BAK1, and SERK4/BKK1—suppressed the phenotypes of an intermediate BRI1 mutant, bri1-5. Overexpression of the kinase-dead versions of these four genes in the bri1-5 background, on the other hand, resulted in typical dominant negative phenotypes, resembling those of null BRI1 mutants. We isolated and generated single, double, triple, and quadruple mutants and analyzed their phenotypes in detail. While the quadruple mutant is embryo-lethal, the serk1 bak1 bkk1 triple null mutant exhibits an extreme de-etiolated phenotype similar to a null bri1 mutant. While overexpression of BRI1 can drastically increase hypocotyl growth of wild-type plants, overexpression of BRI1 does not alter hypocotyl growth of the serk1 bak1 bkk1 triple mutant. Biochemical analysis indicated that the phosphorylation level of BRI1 in serk1 bak1 bkk1 is incapable of sensing exogenously applied BR. As a result, the unphosphorylated level of BES1 has lost its sensitivity to the BR treatment in the triple mutant, indicating that the BR signaling pathway has been completely abolished in the triple mutant. These data clearly demonstrate that SERKs are essential to the early events of BR signaling.
BackgroundTransmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. In Arabidopsis thaliana, there are at least 223 Leucine-rich repeat receptor-like kinases (LRR-RLKs), representing one of the largest protein families. Although functional roles for a handful of LRR-RLKs have been revealed, the functions of the majority of members in this protein family have not been elucidated.ResultsAs a resource for the in-depth analysis of this important protein family, the complementary DNA sequences (cDNAs) of 194 LRR-RLKs were cloned into the GatewayR donor vector pDONR/ZeoR and analyzed by DNA sequencing. Among them, 157 clones showed sequences identical to the predictions in the Arabidopsis sequence resource, TAIR8. The other 37 cDNAs showed gene structures distinct from the predictions of TAIR8, which was mainly caused by alternative splicing of pre-mRNA. Most of the genes have been further cloned into GatewayR destination vectors with GFP or FLAG epitope tags and have been transformed into Arabidopsis for in planta functional analysis. All clones from this study have been submitted to the Arabidopsis Biological Resource Center (ABRC) at Ohio State University for full accessibility by the Arabidopsis research community.ConclusionsMost of the Arabidopsis LRR-RLK genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. The generated resources, including cDNA entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family.
Sequencing results of the rBE14-induced OsWRKY45 mutations in T0 transgenic rice callus lines. (I) Summary of nucleotide changes in the editing window of the endogenous OsWRKY45 gene in T0 transgenic rice callus lines. (J) Summary of base editing efficiencies of rBE14 with different DNA contexts at five genomic sites. (K) Visualizing GFP in transgenic rice callus under a handheld UV lamp and confocal microscope. For (B), (E), (G), (H), and (I), The PAM sequence, the candidate bases in the putative editing window, the detected nucleotide changes/the corresponding amino acids and the sgRNA sequence are highlighted in green, red, blue and bold, respectively. For (C) and (F), the nucleotide changes are underlined in the sequencing chromatograms.
Systemic resistance is induced by pathogens and confers protection against a broad range of pathogens. Recent studies have indicated that salicylic acid (SA) derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile systemic resistance signal in tobacco, Arabidopsis, and potato. However, other experiments indicate that jasmonic acid (JA) is a critical mobile signal. Here, we present evidence suggesting both MeSA and methyl jasmonate (MeJA) are essential for systemic resistance against Tobacco mosaic virus (TMV), possibly acting as the initiating signals for systemic resistance. Foliar application of JA followed by SA triggered the strongest systemic resistance against TMV. Furthermore, we use a virus-induced gene-silencing-based genetics approach to investigate the function of JA and SA biosynthesis or signaling genes in systemic response against TMV infection. Silencing of SA or JA biosynthetic and signaling genes in Nicotiana benthamiana plants increased susceptibility to TMV. Genetic experiments also proved the irreplaceable roles of MeSA and MeJA in systemic resistance response. Systemic resistance was compromised when SA methyl transferase or JA carboxyl methyltransferase, which are required for MeSA and MeJA formation, respectively, were silenced. Moreover, high-performance liquid chromatography-mass spectrometry analysis indicated that JA and MeJA accumulated in phloem exudates of leaves at early stages and SA and MeSA accumulated at later stages, after TMV infection. Our data also indicated that JA and MeJA could regulate MeSA and SA production. Taken together, our results demonstrate that (Me)JA and (Me)SA are required for systemic resistance response against TMV.
The phenomenon of delayed flowering after the application of nitrogen (N) fertilizer has long been known in agriculture, but the detailed molecular basis for this phenomenon is largely unclear. Here we used a modified method of suppression-subtractive hybridization to identify two key factors involved in N-regulated flowering time control in Arabidopsis thaliana, namely ferredoxin-NADP + -oxidoreductase and the blue-light receptor cryptochrome 1 (CRY1). The expression of both genes is induced by low N levels, and their loss-offunction mutants are insensitive to altered N concentration. Low-N conditions increase both NADPH/NADP + and ATP/AMP ratios, which in turn affect adenosine monophosphate-activated protein kinase (AMPK) activity. Moreover, our results show that the AMPK activity and nuclear localization are rhythmic and inversely correlated with nuclear CRY1 protein abundance. Low-N conditions increase but high-N conditions decrease the expression of several key components of the central oscillator (e.g., CCA1, LHY, and TOC1) and the flowering output genes (e.g., GI and CO). Taken together, our results suggest that N signaling functions as a modulator of nuclear CRY1 protein abundance, as well as the input signal for the central circadian clock to interfere with the normal flowering process. T he transition from vegetative to reproductive development is a central event in the plant life cycle, which is coordinately regulated by various endogenous and external cues. In the model dicotyledonous plant species Arabidopsis thaliana, five distinct genetic pathways regulating flowering time have been established: the vernalization pathway, photoperiod pathway, gibberellin acid (GA) pathway, autonomous pathway, and endogenous (age) pathway (1). These pathways ultimately converge to regulate a set of floral integrator genes, FLOWERING LOCUS T (FT) and SUPPRESSOR OF CONSTANS 1 (SOC1), which in turn activate the expression of floral meristem identity genes to trigger the formation of flowers (2-4).Plants use the circadian clock as the timekeeping mechanism to measure day length and to ensure flowering at the proper season (5, 6). As a facultative long-day (LD) plant, Arabidopsis flowers earlier under LD conditions than under short-day (SD) conditions. Forward genetics in A. thaliana have identified the GI-CO-FT hierarchy as the canonical genetic pathway promoting flowering specifically under LD conditions (5,7,8). In this pathway, GI (GIGANTEA) can be considered the output point of the circadian clock to control flowering by regulating CONSTANS (CO) expression in the right phase, which activates expression of FT and TSF (TWIN SISTER OF FT) in the companion cells of the phloem within the vascular tissue (2, 9). FT and TSF proteins act as the long-sought florigens that move from leaves to the apical meristem to induce genes required for reproductive development (2-4). Both GI and CO are regulated by the circadian clock and by light signaling simultaneously and at both transcriptional and posttranscriptional levels, to en...
The target site of pi-d2 gene in rice. (D) Representative Sanger sequencing chromatogram of the rBE5-edited pi-d2 alleles with the target G changed in T0 transgenic rice line #3. (E) Efficiency of rBE5-versus rBE3-mediated gene correction of pi-d2 in T0 transgenic rice lines. (F) The target site of OsFLS2 gene in rice. (G) Representative Sanger sequencing chromatogram of the rBE5-edited OsFLS2 alleles with anticipated C > A conversion in T0 transgenic rice line #21. (H) Efficiency of the rBE5 system in base editing OsFLS2 in T0 transgenic rice lines. (I) Sequencing results of the rBE5-induced OsFLS2 mutations in T0 transgenic rice lines. (J) Construct of the rBE9 system used to induce nucleotide changes in rice transgenic plants. (K) Efficiencies and ratios of allelic mutations caused by the rBE3 and rBE9 systems. (L) Base editing efficiencies of the rBE3 and rBE9 systems at the target C in different sequence context. For (C, F, and I), the PAM sequences, the putative target bases in the activity window, and the detected nucleotide/corresponding amino acids are highlighted in green, red, and blue, respectively. For (D and G), nucleotide mutations are underlined in the sequencing chromatograms.
SUMMARYPlant steroid hormones, brassinosteroids (BRs), play essential roles in modulating cell elongation, vascular differentiation, senescence and stress responses. BRs signal through plasma membrane-localized receptor and other components to modulate the BES1/BZR1 (BRI1-EMS SUPPRESSOR 1/BRASSINAZOLE RESISTANT 1) family of transcription factors that modulate thousands of target genes. Arabodopsis thaliana homeodomain-leucine zipper protein 1 (HAT1), which encodes a homeodomain-leucine zipper (HD-Zip) class II transcription factor, was identified through chromatin immunoprecipitation (ChIP) experiments as a direct target gene of BES1. Loss-of-function and gain-of-function mutants of HAT1 display altered BR responses. HAT1 and its close homolog HAT3 act redundantly, as the double mutant hat1 hat3 displayed a reduced BR response that is stronger than the single mutants alone. Moreover, hat1 hat3 enhanced the phenotype of a weak allele of the BR receptor mutant bri1 and suppressed the phenotype of constitutive BR response mutant bes1-D. These results suggest that HAT1 and HAT3 function to activate BR-mediated growth. Expression levels of several BR-repressed genes are increased in hat1 hat3 and reduced in HAT1OX, suggesting that HAT1 functions to repress the expression of a subset of BR target genes. HAT1 and BES1 bind to a conserved homeodomain binding (HB) site and BR response element (BRRE) respectively, in the promoters of some BR-repressed genes. BES1 and HAT1 interact with each other and cooperate to inhibit BRrepressed gene expression. Furthermore, HAT1 can be phosphorylated and stabilized by GSK3 (GLYCOGEN SYNTHASE KINASE 3)-like kinase BIN2 (BRASSINOSTEROID-INSENSITIVE 2), a well established negative regulator of the BR pathway. Our results thus revealed a previously unknown mechanism by which BR signaling modulates BR-repressed gene expression and coordinates plant growth.
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