Honghui Lin

BAK1 and BKK1 Regulate Brassinosteroid-Dependent Growth and Brassinosteroid-Independent Cell-Death Pathways

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.

Genetic Evidence for an Indispensable Role of Somatic Embryogenesis Receptor Kinases in Brassinosteroid Signaling

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.

Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana

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.

Somatic Embryogenesis Receptor Kinases Control Root Development Mainly via Brassinosteroid‐Independent Actions in Arabidopsis thaliana

Brassinosteroids (BRs), a group of plant steroidal hormones, play critical roles in many aspects of plant growth and development. Previous studies showed that BRI1-mediated BR signaling regulates cell division and differentiation during Arabidopsis root development via interplaying with auxin and other phytohormones. Arabidopsis somatic embryogenesis receptor-like kinases (SERKs), as co-receptors of BRI1, were found to play a fundamental role in an early activation step of BR signaling pathway. Here we report a novel function of SERKs in regulating Arabidopsis root development. Genetic analyses indicated that SERKs control root growth mainly via a BR-independent pathway. Although BR signaling pathway is completely disrupted in the serk1 bak1 bkk1 triple mutant, the root growth of the triple mutant is much severely damaged than the BR deficiency or signaling null mutants. More detailed analyses indicated that the triple mutant exhibited drastically reduced expression of a number of genes critical to polar auxin transport, cell cycle, endodermis development and root meristem differentiation, which were not observed in null BR biosynthesis mutant cpd and null BR signaling mutant bri1-701.

Transcription factor HAT1 is phosphorylated by BIN2 kinase and mediates brassinosteroid repressed gene expression in Arabidopsis.

Plant 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 BR-repressed 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.

Transcription factor HAT1 is a substrate of SnRK2.3 kinase and negatively regulates ABA synthesis and signaling in Arabidopsis responding to drought

Drought is a major threat to plant growth and crop productivity. The phytohormone abscisic acid (ABA) plays a critical role in plant response to drought stress. Although ABA signaling-mediated drought tolerance has been widely investigated in Arabidopsis thaliana, the feedback mechanism and components negatively regulating this pathway are less well understood. Here we identified a member of Arabidopsis HD-ZIP transcription factors HAT1 which can interacts with and be phosphorylated by SnRK2s. hat1hat3, loss-of-function mutant of HAT1 and its homolog HAT3, was hypersensitive to ABA in primary root inhibition, ABA-responsive genes expression, and displayed enhanced drought tolerance, whereas HAT1 overexpressing lines were hyposensitive to ABA and less tolerant to drought stress, suggesting that HAT1 functions as a negative regulator in ABA signaling-mediated drought response. Furthermore, expression levels of ABA biosynthesis genes ABA3 and NCED3 were repressed by HAT1 directly binding to their promoters, resulting in the ABA level was increased in hat1hat3 and reduced in HAT1OX lines. Further evidence showed that both protein stability and binding activity of HAT1 was repressed by SnRK2.3 phosphorylation. Overexpressing SnRK2.3 in HAT1OX transgenic plant made a reduced HAT1 protein level and suppressed the HAT1OX phenotypes in ABA and drought response. Our results thus establish a new negative regulation mechanism of HAT1 which helps plants fine-tune their drought responses.