Tae-Houn Kim

Guard Cell Signal Transduction Network: Advances in Understanding Abscisic Acid, CO2, and Ca2+ Signaling

Stomatal pores are formed by pairs of specialized epidermal guard cells and serve as major gateways for both CO(2) influx into plants from the atmosphere and transpirational water loss of plants. Because they regulate stomatal pore apertures via integration of both endogenous hormonal stimuli and environmental signals, guard cells have been highly developed as a model system to dissect the dynamics and mechanisms of plant-cell signaling. The stress hormone ABA and elevated levels of CO(2) activate complex signaling pathways in guard cells that are mediated by kinases/phosphatases, secondary messengers, and ion channel regulation. Recent research in guard cells has led to a new hypothesis for how plants achieve specificity in intracellular calcium signaling: CO(2) and ABA enhance (prime) the calcium sensitivity of downstream calcium-signaling mechanisms. Recent progress in identification of early stomatal signaling components are reviewed here, including ABA receptors and CO(2)-binding response proteins, as well as systems approaches that advance our understanding of guard cell-signaling mechanisms.

Triple Loss of Function of Protein Phosphatases Type 2C Leads to Partial Constitutive Response to Endogenous Abscisic Acid

The phytohormone abscisic acid (ABA) is a key regulator of plant growth and development as well as plant responses to situations of decreased water availability. Protein phosphatases type 2C (PP2Cs) from group A, which includes the ABI1/HAB1 and PP2CA branches, are key negative regulators of ABA signaling. Specifically, HAB1, ABI1, ABI2, and PP2CA have been shown to affect both seed and vegetative responses to ABA. To further understand their contribution to ABA signaling and to unravel possible genetic interactions and functional redundancy among them, we have generated different combinations of double and triple mutants impaired in these PP2Cs. Interestingly, hab1-1pp2ca-1 and abi1-2pp2ca-1 double mutants showed reduced water loss and enhanced resistance to drought stress, which further supports the role of PP2CA in vegetative responses to ABA. Two triple hab1-1abi1-2abi2-2 and hab1-1abi1-2pp2ca-1 mutants were generated, which showed an extreme response to exogenous ABA, impaired growth, and partial constitutive response to endogenous ABA. Thus, transcriptomic analysis revealed a partial up-regulation/down-regulation of a subset of ABA-responsive genes in both triple mutants in the absence of exogenous ABA. Comparison of ABA responses in the different pp2c mutants showed that a progressive increase in ABA sensitivity could be obtained through combined inactivation of these PP2Cs. These results indicate that ABA response is finely tuned by the integrated action of these genes, which is required to prevent a constitutive response to endogenous ABA that might have a deleterious effect on growth and development in the absence of environmental stress.

The Peroxin Loss-of-Function Mutation abstinence by mutual consent Disrupts Male-Female Gametophyte Recognition

In eukaryotes, fertilization relies on complex and specialized mechanisms that achieve the precise delivery of the male gamete to the female gamete and their subsequent union [1-4]. In flowering plants, the haploid male gametophyte or pollen tube (PT) [5] carries two nonmotile sperm cells to the female gametophyte (FG) or embryo sac [6] during a long assisted journey through the maternal tissues [7-10]. In Arabidopsis, typically one PT reaches one of the two synergids of the FG (Figure 1A), where it terminates its growth and delivers the sperm cells, a poorly understood process called pollen-tube reception. Here, we report the isolation and characterization of the Arabidopsis mutant abstinence by mutual consent (amc). Interestingly, pollen-tube reception is impaired only when an amc pollen tube reaches an amc female gametophyte, resulting in pollen-tube overgrowth and completely preventing sperm discharge and the development of homozygous mutants. Moreover, we show that AMC is strongly and transiently expressed in both male and female gametophytes during fertilization and that AMC functions in gametophytes as a peroxin essential for protein import into peroxisomes. These findings show that peroxisomes play an unexpected key role in gametophyte recognition and implicate a diffusible signal emanating from either gametophyte that is required for pollen-tube discharge.

PCI complexes: pretty complex interactions in diverse signaling pathways

Three protein complexes (the proteasome regulatory lid, the COP9 signalosome and eukaryotic translation initiation factor 3) contain protein subunits with a well defined protein domain, the PCI domain. At least two (the COP9 signalosome and the lid) appear to share a common evolutionary origin. Recent advances in our understanding of the structure and function of the three complexes point to intriguing and unanticipated connections between the cellular functions performed by these three protein assemblies, especially between translation initiation and proteolytic protein degradation.

Translational Regulation via 5′ mRNA Leader Sequences Revealed by Mutational Analysis of the Arabidopsis Translation Initiation Factor Subunit eIF3h

Eukaryotic translation initiation factor 3 (eIF3) consists of core subunits that are conserved from yeast to man as well as less conserved, noncore, subunits with potential regulatory roles. Whereas core subunits tend to be indispensable for cell growth, the roles of the noncore subunits remain poorly understood. We addressed the hypothesis that eIF3 noncore subunits have accessory functions that help to regulate translation initiation, by focusing on the Arabidopsis thaliana eIF3h subunit. Indeed, eIF3h was not essential for general protein translation. However, results from transient expression assays and polysome fractionation indicated that the translation efficiency of specific 5′ mRNA leader sequences was compromised in an eif3h mutant, including the mRNA for the basic domain leucine zipper (bZip) transcription factor ATB2/AtbZip11, translation of which is regulated by sucrose. Among other pleiotropic developmental defects, the eif3h mutant required exogenous sugar to transit from seedling to vegetative development, but it was hypersensitive to elevated levels of exogenous sugars. The ATB2 mRNA was rendered sensitive to the eIF3h level by a series of upstream open reading frames. Moreover, eIF3h could physically interact with subunits of the COP9 signalosome, a protein complex implicated primarily in the regulation of protein ubiquitination, supporting a direct biochemical connection between translation initiation and protein turnover. Together, these data implicate eIF3 in mRNA-associated translation initiation events, such as scanning, start codon recognition, or reinitiation and suggest that poor translation initiation of specific mRNAs contributes to the pleiotropic spectrum of phenotypic defects in the eif3h mutant.

Arabidopsis eIF3e (INT-6) Associates with Both eIF3c and the COP9 Signalosome Subunit CSN7

The Arabidopsis COP9 signalosome is a multisubunit repressor of photomorphogenesis that is conserved among eukaryotes. This complex may have a general role in development. As a step in dissecting the biochemical mode of action of the COP9 signalosome, we determined the sequence of proteins that copurify with this complex. Here we describe the association between components of the COP9 signalosome (CSN1, CSN7, and CSN8) and two subunits of eukaryotic translation initiation factor 3 (eIF3), eIF3e (p48, known also as INT-6) and eIF3c (p105). To obtain a biochemical marker forArabidopsis eIF3, we cloned the Arabidopsisortholog of the eIF3 subunit eIF3b (PRT1). eIF3e coimmunoprecipitated with CSN7, and eIF3c coimmunoprecipitated with eIF3e, eIF3b, CSN8, and CSN1. eIF3e directly interacted with CSN7 and eIF3c. However, eIF3e and eIF3b cofractionated by gel filtration chromatography in a complex that was larger than the COP9 signalosome. Whereas eIF3, as detected through eIF3b, localized solely to the cytoplasm, eIF3e, like CSN7, was also found in the nucleus. This suggests that eIF3e and eIF3c are probably components of multiple complexes and that eIF3e and eIF3c associate with subunits of the COP9 signalosome, even though they are not components of the COP9 signalosome core complex. This interaction may allow for translational control by the COP9 signalosome.

ARS5 is a component of the 26S proteasome complex and negatively regulates thiol biosynthesis and arsenic tolerance in Arabidopsis

A forward-genetic screen in Arabidopsis led to the isolation of several arsenic tolerance mutants. ars5 was the strongest arsenate- and arsenite-resistant mutant identified in this genetic screen. Here, we report the characterization and cloning of the ars5 mutant gene. ars5 is shown to exhibit an increased accumulation of arsenic and thiol compounds during arsenic stress. Rough mapping together with microarray-based expression mapping identified the ars5 mutation in the alpha subunit F (PAF1) of the 26S proteasome complex. Characterization of an independent paf1 T-DNA insertion allele and complementation by PAF1 confirmed that paf1 mutation is responsible for the enhanced thiol accumulation and arsenic tolerance phenotypes. Arsenic tolerance was not observed in a knock-out mutant of the highly homologous PAF2 gene. However, genetic complementation of ars5 by the overexpression of PAF2 suggests that the PAF2 protein is functionally equivalent to PAF1 when expressed at high levels. No detectible difference was observed in total ubiquitinylated protein profiles between ars5 and wild-type (WT) Arabidopsis, suggesting that the arsenic tolerance observed in ars5 is not derived from a general impairment in proteasome-mediated protein degradation. Quantitative RT-PCR showed that arsenic induces the enhanced transcriptional activation of several key genes that function in glutathione and phytochelatin biosynthesis in the WT, and this arsenic induction of gene expression is more dramatic in ars5. The enhanced transcriptional response to arsenic and the increased accumulation of thiol compounds in ars5, compared with WT, suggest the presence of a positive regulation pathway for thiol biosynthesis that is enhanced in the ars5 background.

Natural variation in small molecule-induced TIR-NB-LRR signaling induces root growth arrest via EDS1- and PAD4-complexed R protein VICTR in Arabidopsis.

This chemical genetics work reveals natural variation in a newly identified R protein homolog, named VICTR, that produces primary root growth arrest in response to the small molecule DFPM. DFPM perception and signal transduction require early components of the plant R gene resistance signaling network, and the R protein VICTR coresides in complexes not only with EDS1 but also PAD4. In a chemical genetics screen we identified the small-molecule [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) that triggers rapid inhibition of early abscisic acid signal transduction via PHYTOALEXIN DEFICIENT4 (PAD4)- and ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent immune signaling mechanisms. However, mechanisms upstream of EDS1 and PAD4 in DFPM-mediated signaling remain unknown. Here, we report that DFPM generates an Arabidopsis thaliana accession-specific root growth arrest in Columbia-0 (Col-0) plants. The genetic locus responsible for this natural variant, VICTR (VARIATION IN COMPOUND TRIGGERED ROOT growth response), encodes a TIR-NB-LRR (for Toll-Interleukin1 Receptor–nucleotide binding–Leucine-rich repeat) protein. Analyses of T-DNA insertion victr alleles showed that VICTR is necessary for DFPM-induced root growth arrest and inhibition of abscisic acid–induced stomatal closing. Transgenic expression of the Col-0 VICTR allele in DFPM-insensitive Arabidopsis accessions recapitulated the DFPM-induced root growth arrest. EDS1 and PAD4, both central regulators of basal resistance and effector-triggered immunity, as well as HSP90 chaperones and their cochaperones RAR1 and SGT1B, are required for the DFPM-induced root growth arrest. Salicylic acid and jasmonic acid signaling pathway components are dispensable. We further demonstrate that VICTR associates with EDS1 and PAD4 in a nuclear protein complex. These findings show a previously unexplored association between a TIR-NB-LRR protein and PAD4 and identify functions of plant immune signaling components in the regulation of root meristematic zone-targeted growth arrest.