[Show abstract][Hide abstract]ABSTRACT: RGF1, a secreted peptide hormone, plays key roles in root meristem development in Arabidopsis. Previous studies indicated that a functional RGF1 needs to be sulfated at a tyrosine residue by a tyrosylprotein sulfotransferase and that RGF1 regulates the root meristem activity mainly via two downstream transcription factors, PLETHORA 1 (PLT1) and PLT2. How extracellular RGF1 is perceived by a plant cell, however, is unclear. Using genetic approaches, we discovered a clade of leucine-rich repeat receptor-like kinases, designated as RGF1 INSENSITIVE 1 (RGI1) to RGI5, serving as receptors of RGF1. Two independent rgi1 rgi2 rgi3 rgi4 rgi5 quintuple mutants display a consistent short primary root phenotype with a small size of meristem. An rgi1 rgi2 rgi3 rgi4 quadruple mutant shows a significantly reduced sensitivity to RGF1, and the quintuple mutant is completely insensitive to RGF1. The expression of PLT1 and PLT2 is almost undetectable in the quintuple mutant. Ectopic expression of PLT2 driven by an RGI2 promoter in the quintuple mutant greatly rescued its root meristem defects. One of the RGIs, RGI1, was subsequently analyzed biochemically in detail. In vitro dot blotting and pull-down analyses indicated that RGI1 can physically interact with RGF1. Exogenous application of RGF1 can quickly and simultaneously induce the phosphorylation and ubiquitination of RGI1, indicating that RGI1 can perceive and transduce the RGF1 peptide signal. Yet, the activated RGI1 is likely turned over rapidly. These results demonstrate that RGIs, acting as the receptors of RGF1, play essential roles in RGF1-PLT-mediated root meristem development in Arabidopsis thaliana.Cell Research advance online publication 27 May 2016; doi:10.1038/cr.2016.63.
[Show abstract][Hide abstract]ABSTRACT: Stomata are highly specialized epidermal structures that control transpiration and gas exchange between plants and the environment. Signal networks underlying stomatal development have been previously uncovered, however, much less is known about how signals involved in stomatal development are transmitted to RNA polymerase II (Pol II), which plays a central role in the transcription of mRNA coding genes. Herein, we identified a partial loss-of-function mutation of the third largest subunit of nuclear DNA-dependent RNA polymerase II (NRPB3) that exhibited an increased number of stomatal lineage cells and paired stomata. Phenotypic and genetic analyses indicated that NRPB3 was not only required for correct stomatal patterning, but was also essential for stomatal differentiation. Protein-protein interaction assays showed that NRPB3 directly interacted with two basic-helix-loop-helix (bHLH) transcription factors, FAMA and INDUCER OF CBF EXPRESSION1 (ICE1), indicating that NRPB3 serves as an acceptor for signals from transcription factors involved in stomatal development. Our findings highlight the surprisingly conserved activating mechanisms mediated by the third largest subunit of Pol II in eukaryotes.
[Show abstract][Hide abstract]ABSTRACT: BAK1 was initially identified as a co-receptor of the brassinosteroid (BR) receptor, BRI1. Genetic analyses also revealed that BAK1 and its closest homolog BKK1 regulate a BR-independent cell-death control pathway. A double null mutant bak1 bkk1 displays a salicylic acid (SA)- and light-dependent cell-death phenotype even without pathogen invasion. Molecular mechanisms of the spontaneous cell death mediated by BAK1 and BKK1 remain elusive. Here we report our identification of a suppressor of bak1 bkk1 (sbb1-1). Genetic analyses indicated that the cell-death symptom in a weak double mutant, bak1-3 bkk1-1, was completely suppressed by the loss-of-function mutation of SBB1, which encodes a nucleoporin (NUP) 85-like protein. Genetic analyses also demonstrated that knocking-out three other NUPs individually from the SBB1-located sub-complex was also able to rescue the cell-death phenotype of bak1-3 bkk1-1. In addition, a DEAD box RNA helicase, DRH1, was identified in the same protein complex of SBB1 via a proteomic approach. The drh1 mutant can also rescue the cell-death symptom of bak1-3 bkk1-1. Further analyses indicated that the export of poly (A)(+) RNA was greatly blocked, resulting in accumulation of significant amount of mRNAs in the nuclei of the nup and drh1 mutants. Overexpression of a bacterial NahG gene to inactivate SA can rescue the cell-death symptom of bak1-3 bkk1-1. Meanwhile, mutants suppressing the cell death symptoms always showed greatly reduced SA contents. These results suggest that nucleocytoplasmic trafficking, especially for the molecules directly or indirectly involved in endogenous SA accumulation, is critical in BAK1 and BKK1-mediated cell-death control. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract]ABSTRACT: Flowering plant sperm cells transcribe a divergent and complex complement of genes. To examine promoter function, we chose an isopentenyltransferase gene known as PzIPT1. This gene is highly selectively transcribed in one sperm cell morphotype (Svn) of Plumbago zeylanica, which preferentially fuses with the central cell during fertilization and is thus a founding cell of the primary endosperm. In transgenic Arabidopsis, PzIPT1 promoter displays activity in both sperm cells and upon progressive promoter truncation from the 5'-end results in a progressive decrease in reporter production, consistent with occurrence of multiple enhancer sites. Cytokinin-dependent protein binding (CPB) motifs are identified in the promoter sequence, which respond with stimulation by cytokinin. Expression of PzIPT1 promoter in sperm cells confers specificity independently of previously reported Germline Restrictive Silencer Factor (GRSF) binding sequence. Instead, a cis-acting regulatory region consisting of two duplicated 6-bp Male Gamete Selective Activation (MGSA) motifs occurs near the site of transcription initiation. Disruption of this sequence-specific site inactivates expression of a GFP reporter gene in sperm cells. Multiple copies of the MGSA motif fused with the minimal CaMV35S promoter elements confer reporter gene expression in sperm cells. Similar duplicated MGSA motifs are also identified from promoter sequences of sperm cell-expressed genes in Arabidopsis, suggesting selective activation is possibly a common mechanism for regulation of gene expression in sperm cells of flowering plants.
[Show abstract][Hide abstract]ABSTRACT: In plants, photoreceptors transfer light signals to phytochrome (phy)-interacting factors (PIFs), inducing PIFs rapid phosphorylation and degradation to promote photomorphogenesis. However, the phosphatase responsible for PIF dephosphorylation remains unknown. In this study, we identified a type-one protein phosphatase, TOPP4 that is essential for PIF5 protein stability in Arabidopsis. Compared to the wild type, the dominant-negative mutant, topp4-1, displayed reduced hypocotyl length and larger apical hook and cotyledon opening angle under red light. Overexpression of topp4-1 in the wild type led to defects that were similar to those in topp4-1 mutant. Red light induced phyB-dependent TOPP4 expression in hypocotyls. The topp4-1 mutation weakened the closed cotyledon angle of phyB-9 and phyA-211 phyB-9, while overexpression of TOPP4 significantly repressed the short hypocotyls of phyB-GFP seedlings, indicating that TOPP4 and phyB function in an antagonistic way during photomorphogenesis. Protein interaction assays and phosphorylation studies demonstrate that TOPP4 directly interacts with PIF5 and dephosphorylates it. Furthermore, TOPP4 inhibits red-light induced ubiquitination and degradation of PIF5. These findings demonstrate that dephosphorylation of PIF5 by TOPP4 inhibits its ubiquitin-mediated degradation during photomorphogenesis. These data outline a novel phytochrome signaling mechanism that TOPP4-mediated dephosphorylation of PIF5 attenuates phytochrome-dependent light responses.
[Show abstract][Hide abstract]ABSTRACT: Receptor-like protein kinases (RLKs) are a large group of transmembrane proteins playing critical roles in cell-cell and cell-environment communications. Based on extracellular domain structures, RLKs were classified into more than 21 subfamilies, among which leucine-rich repeat RLKs (LRR-RLKs) belong to the largest subfamily in plants such as Arabidopsis and rice. In Arabidopsis, there are approximately 223 LRR-RLKs. Only about 60 of them have been functionally described. The majority of them need to be functionally determined. To systematically investigate their roles in regulating plant growth, development and stress adaptations, we generated promoter::GUS transgenic plants for all 223 LRR-RLKs and analyzed their detailed expression patterns at various developmental stages. These results provide valuable resources for the research community to functionally elucidate this large and essential signaling protein subfamily.
[Show abstract][Hide abstract]ABSTRACT: Traits related to grain and reproductive organs in grass crops have been under continuous directional selection during domestication. Barley is one of the oldest domesticated crops in human history. Thus genes associated with the grain and reproductive organs in barley may show evidence of dramatic evolutionary change. To understand how artificial selection contributes to protein evolution of biased genes in different barley organs, we used Digital Gene Expression analysis of six barley organs (grain, pistil, anther, leaf, stem and root) to identify genes with biased expression in specific organs. Pairwise comparisons of orthologs between barley and Brachypodium distachyon, as well as between highland and lowland barley cultivars mutually indicated that grain and pistil biased genes show relatively higher protein evolutionary rates compared with the median of all orthologs and other organ biased genes. Lineage-specific protein evolutionary rates estimation showed similar patterns with elevated protein evolution in barley grain and pistil biased genes, yet protein sequences generally evolve much faster in the lowland barley cultivar. Further functional annotations revealed that some of these grain and pistil biased genes with rapid protein evolution are related to nutrient biosynthesis and cell cycle/division. Our analyses provide insights into how domestication differentially shaped the evolution of genes specific to different organs of a crop species, and implications for future functional studies of domestication genes.
[Show abstract][Hide abstract]ABSTRACT: The formation of a zygote by the fusion of egg and sperm involves the two gametic transcriptomes. In flowering plants, the embryo sac embedded within the ovule contains the egg cell, whereas the pollen grain contains two sperm cells inside a supporting vegetative cell. The difficulties of collecting isolated gametes and consequent low recovery of RNA have restricted in-depth analysis of gametic transcriptomes in flowering plants. We isolated living egg cells, sperm cells and pollen vegetative cells from Oryza sativa (rice), and identified transcripts for approximately 36 000 genes by deep sequencing. The three transcriptomes are highly divergent, with about three-quarters of those genes differentially expressed in the different cell types. Distinctive expression profiles were observed for genes involved in chromatin conformation, including an unexpected expression in the sperm cell of genes associated with active chromatin. Furthermore, both the sperm cell and the pollen vegetative cell were deficient in expression of key RNAi components. Differences in gene expression were also observed for genes for hormonal signaling and cell cycle regulation. The egg cell and sperm cell transcriptomes reveal major differences in gene expression to be resolved in the zygote, including pathways affecting chromatin configuration, hormones and cell cycle. The sex-specific differences in the expression of RNAi components suggest that epigenetic silencing in the zygote might act predominantly through female-dependent pathways. More generally, this study provides a detailed gene expression landscape for flowering plant gametes, enabling the identification of specific gametic functions, and their contributions to zygote and seed development.
[Show abstract][Hide abstract]ABSTRACT: Genomic assay of sperm cell RNA provides insight into functional control, modes of regulation, and contributions of male gametes to double fertilization. Sperm cells of rice (Oryza sativa) were isolated from field-grown, disease-free plants and RNA was processed for use with the full-genome Affymetrix microarray. Comparison with Gene Expression Omnibus (GEO) reference arrays confirmed expressionally distinct gene profiles. A total of 10,732 distinct gene sequences were detected in sperm cells, of which 1668 were not expressed in pollen or seedlings. Pathways enriched in male germ cells included ubiquitin-mediated pathways, pathways involved in chromatin modeling including histones, histone modification and nonhistone epigenetic modification, and pathways related to RNAi and gene silencing. Genome-wide expression patterns in angiosperm sperm cells indicate common and divergent themes in the male germline that appear to be largely self-regulating through highly up-regulated chromatin modification pathways. A core of highly conserved genes appear common to all sperm cells, but evidence is still emerging that another class of genes have diverged in expression between monocots and dicots since their divergence. Sperm cell transcripts present at fusion may be transmitted through plasmogamy during double fertilization to effect immediate post-fertilization expression of early embryo and (or) endosperm development.
[Show abstract][Hide abstract]ABSTRACT: 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.
[Show abstract][Hide abstract]ABSTRACT: Representative loss-of-function mutant phenotypes of the mutants generated by an independent set of T-DNA insertion null mutants of SERKs. A. Representative loss-of-function phenotypes of 28-day-old SERK mutants in the light. Only bak1-6 shows weak bri1-like phenotypes among the single knock-out mutants with smaller rosette size. The double knock-out mutant serk1-1 bak1-6 shows similar phenotypes as the bri1 weak allele bri1-301, and bak1-6 bkk1-2 shows a seedling-lethality phenotype at the early developmental stage. The triple knock-out mutant serk1-1 bak1-6 bkk1-2 shows similar seedling lethality phenotypes as the bak1-6 bkk1-2 mutant plants. B. Representative loss-of-function phenotypes of 5-day-old SERK mutants in the dark. bri1-701 shows a typical null bri1 phenotype in the dark with opened cotyledons, shortened and swollen hypocotyls. Double null mutant serk1-1 bak1-6 shows a similar phenotype to the bri1-701, with longer hypocotyls. The triple knock-out mutant serk1-1 bak1-6 bkk1-2 shows similar phenotypes to the bri1-701, with completely opened cotyledons, shortened and swollen hypocotyls. C. Measurements of the dark-grown seedlings shown in B. Error bars represent SD.
[Show abstract][Hide abstract]ABSTRACT: bak1-3 is a leaky T-DNA insertion mutant in bak1-3 bkk1-1 background, whereas bak1-4 is likely a null mutant. A. T-DNA insertion sites for bak1-3 and bak1-4. Filled boxes represent exons, lines between boxes represent introns. The positions of the used primers are shown with arrows and the sequences are listed in Table S1. F: BAK1F; R: BAK1R; F1: BAK1F1; R1: BAK1R1; F2: BAK1F2; R2: BAK1R2. B. RT-PCR analyses indicated that bak1-3 is a leaky mutant in bkk1-1 background. In bak1-3 bkk1-1 double but not in bak1-3 single mutant background, wild-type BAK1 cDNA can still be detected by RT-PCR. But there is no wild-type like full-length BAK1 cDNA can be detected by RT-PCR in bak1-4 single or bak1-4 bkk1-1 double mutants. The used primer pairs are shown at the right. C. Phenotypes of 19-day-old wild-type, bak1-3, bak1-4, bak1-3 bkk1-1, and bak1-4 bkk1-1. bak1-3 single mutant shows phenotypic defects similar to bak1-4; whereas bak1-3 bkk1-1 double mutant shows a much milder phenotype than bak1-4 bkk1-1.
[Show abstract][Hide abstract]ABSTRACT: BRI1 and SERK mutants used in these studies do not express full-length mRNA. A. Expression of BRI1 in bri1-701 plants. RT-PCR reactions were performed to detect the full-length CDS (upper) and the mRNA sequence flanking the T-DNA insertion site (middle) in the wild type and the bir1-701 mutant. The primer pairs used are indicated at the right. ACTIN2 was amplified as a control (Lower). B, C. Expression of SERKs in serk mutant plants. The full-length CDS sequences and the mRNA sequences flanking T-DNA insertion sites were amplified with primer pairs listed in Table S1. The mutants are indicated on the top.
[Show abstract][Hide abstract]ABSTRACT: 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.
[Show abstract][Hide abstract]ABSTRACT: SERK2 shows a basal level of interaction with BRI1. Co-immunoprecipitation result indicates that SERK2 can interact with BRI1 at a minimal level. The interaction cannot be enhanced by the exogenously applied BR. The phosphorylation of SERK2, on the other hand, is elevated to BR treatment.
[Show abstract][Hide abstract]ABSTRACT: BES1 phosphorylation levels are not responsive to BR in the triple mutant serk1-1 bak1-6 bkk1-2 generated by the 2nd independent set of T-DNA insertion lines. A. Seven-day-old seedlings of wild-type and mutants grown in the light were treated with 0 or 1 µM 24-epiBL for 4 h. Total proteins were analyzed by Western hybridization with a specific anti-BES1 antibody. BES1 response upon BR treatment in the triple mutant serk1-1 bak1-6 bkk1-2 is blocked. B. Coomassie blue staining of PAGE-separated proteins to show equally loaded proteins between each pair of treated and untreated samples. BL, BR treatment. −, without BR treatment; +, with BR treatment. BES1-P, phosphorylated BES1; BES1, unphosphorylated BES1.