Molecular Plant - recent issues

Transcomplementation, but not Physical Association of the CC-NB-ARC and LRR Domains of Tomato R Protein Mi-1.2 is Altered by Mutations in the ARC2 Subdomain

van Ooijen, G., Mayr, G., Albrecht, M., Cornelissen, B. J. C., Takken, F. L.W. — 2008-05-29

Race-specific disease resistance in plants is mediated by Resistance (R) proteins that recognize pathogen attack and initiate defence responses. Most R proteins contain a central NB-ARC domain and a C-terminal leucine-rich repeat (LRR) domain. We analyzed the intramolecular interaction of the LRR domain of tomato R protein Mi-1.2 with its N-terminus. We expressed the CC-NB-ARC and LRR parts in trans and analyzed functional transcomplementation and physical interactions. We show that these domains functionally transcomplement when expressed in trans. Known autoactivating LRR domain swaps were found to induce a hypersensitive response (HR) upon co-expression. Likewise, autoactivating mutants in the NB subdomain transcomplemented to induce HR. Point mutations in the ARC2 subdomain that induce strong autoactivation in the full-length Mi-1.2 protein, however, fail to induce HR in the transcomplementation assay. These data indicate distinct functions for the NB-ARC subdomains in induction of HR signalling. Furthermore, dissociation of the LRR is not required to release its negative regulation, as in all combinations of CC-NB-ARC and LRR domains tested, a physical interaction was observed.

Nuclear Trafficking During Plant Innate Immunity

Liu, J., Coaker, G. — 2008-05-29

Land plants possess innate immune systems that can control resistance against pathogen infection. Conceptually, there are two branches of the plant innate immune system. One branch recognizes conserved features of microbial pathogens, while a second branch specifically detects the presence of pathogen effector proteins by plant resistance (R) genes. Innate immunity controlled by plant R genes is called effector-triggered immunity. Although R genes can recognize all classes of plant pathogens, the majority can be grouped into one large family, encoding proteins with a nucleotide binding site and C-terminal leucine rich repeat domains. Despite the importance and number of R genes present in plants, we are just beginning to decipher the signaling events required to initiate defense responses. Recent exciting discoveries have implicated dynamic nuclear trafficking of plant R proteins to achieve effector-triggered immunity. Furthermore, there are several additional lines of evidence implicating nucleo-cyctoplasmic trafficking in plant disease resistance, as mutations in nucleoporins and importins can compromise resistance signaling. Taken together, these data illustrate the importance of nuclear trafficking in the manifestation of disease resistance mediated by R genes.

Activation of Defense Response Pathways by OGs and Flg22 Elicitors in Arabidopsis Seedlings

2008-05-29

We carried out transcriptional profiling analysis in 10-d-old Arabidopsis thaliana seedlings treated with oligogalacturonides (OGs), oligosaccharides derived from the plant cell wall, or the bacterial flagellin peptide Flg22, general elicitors of the basal defense response in plants. Although detected by different receptors, both OGs and Flg22 trigger a fast and transient response that is both similar and comprehensive, and characterized by activation of early stages of multiple defense signaling pathways, particularly JA-associated processes. However, the response to Flg22 is stronger in both the number of genes differentially expressed and the amplitude of change. The magnitude of induction of individual genes is in both cases dose-dependent, but, even at very high concentrations, OGs do not induce a response that is as comprehensive as that seen with Flg22. While high doses of either microbe-associated molecular pattern (MAMP) elicit a late response that includes activation of senescence processes, SA-dependent secretory pathway genes and PR1 expression are substantially induced only by Flg22. These results suggest a lower threshold for activation of early responses than for sustained or SA-mediated late defenses. Expression patterns of amino–cyclopropane–carboxylate synthase genes also implicate ethylene biosynthesis in regulation of the late innate immune response.

OsWRKY62 is a Negative Regulator of Basal and Xa21-Mediated Defense against Xanthomonas oryzae pv. oryzae in Rice

2008-05-29

The rice Xa21 gene, which confers resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), encodes a receptor-like kinase. Few components involved in transducing the Xa21-mediated defense response have yet been identified. Here, we report that XA21 binds to a WRKY transcription factor, called OsWRKY62. The OsWRKY62 gene encodes two splice variants (OsWRKY62.1 and OsWRKY62.2). OsWRKY62.1:smGFP2 and OsWRKY62.2:smGFP2 fusion proteins partially localize to the nucleus. Transgenic plants overexpressing OsWRKY62.1 are compromised in basal defense and Xa21-mediated resistance to Xoo. Furthermore, overexpression of OsWRKY62.1 suppresses the activation of defense-related genes. These results imply that OsWRKY62 functions as a negative regulator of innate immunity in rice, and serves as a critical mediator of both basal and race-specific defense responses.

Stress- and Pathogen-Induced Arabidopsis WRKY48 is a Transcriptional Activator that Represses Plant Basal Defense

Xing, D.-H., Lai, Z.-B., Zheng, Z.-Y., Vinod, K. M., Fan, B.-F., Chen, Z.-X. — 2008-05-29

Plant WRKY transcription factors can function as either positive or negative regulators of plant basal disease resistance. Arabidopsis WRKY48 is induced by mechanical and/or osmotic stress due to infiltration and pathogen infection and, therefore, may play a role in plant defense responses. WRKY48 is localized to the nucleus, recognizes the TTGACC W-box sequence with a high affinity in vitro and functions in plant cells as a strong transcriptional activator. To determine the biological functions directly, we have isolated loss-of-function T-DNA insertion mutants and generated gain-of-function transgenic overexpression plants for WRKY48 in Arabidopsis. Growth of a virulent strain of the bacterial pathogen Pseudomonas syringae was decreased in the wrky48 T-DNA insertion mutants. The enhanced resistance of the loss-of-function mutants was associated with increased induction of salicylic acid-regulated PR1 by the bacterial pathogen. By contrast, transgenic WRKY48-overexpressing plants support enhanced growth of P. syringae and the enhanced susceptibility was associated with reduced expression of defense-related PR genes. These results suggest that WRKY48 is a negative regulator of PR gene expression and basal resistance to the bacterial pathogen P. syringae.

Mutation of a Gene in the Fungus Leptosphaeria maculans Allows Increased Frequency of Penetration of Stomatal Apertures of Arabidopsis thaliana

Elliott, C. E., Harjono, , Howlett, B. J. — 2008-05-29

Leptosphaeria maculans, a pathogen of Brassica napus, is unable to invade most wild-type accessions of Arabidopsis thaliana, although several mutants are susceptible. The infection pathway of L. maculans via a non-invasive inoculation method on A. thaliana lms1 (undefined), pmr4-1 (defective in callose deposition), and pen1-1 and pen2-1 (defective in non-host responses to several pathogens) mutants is described. On wild types Col-0 and Ler-0, hyphae are generally arrested at stomatal apertures. A T-DNA insertional mutant of L. maculans (A22) that penetrates stomatal apertures of Col-0 and Ler-0 five to seven times more often than the wild-type isolate is described. The higher penetration frequency of isolate A22 is associated with an increased hypersensitive response, which includes callose deposition. Complementation analysis showed that the phenotype of this isolate is due to T-DNA insertion in an intronless gene denoted as ipa (increased penetration on Arabidopsis). This gene is predicted to encode a protein of 702 amino acids with best matches to hypothetical proteins in other filamentous ascomycetes. The ipa gene is expressed in the wild-type isolate at low levels in culture and during infection of A. thaliana and B. napus.

Kunitz Trypsin Inhibitor: An Antagonist of Cell Death Triggered by Phytopathogens and Fumonisin B1 in Arabidopsis

Li, J., Brader, G., Palva, E. T. — 2008-05-29

Programmed cell death (PCD) is a central regulatory process in both plant development and in plant responses to pathogens. PCD requires a coordinate activation of pro-apoptotic factors such as proteases and suppressors inhibiting and modulating these processes. In plants, various caspase-like cysteine proteases as well as serine proteases have been implicated in PCD. Here, we show that a serine protease (Kunitz trypsin) inhibitor (KTI1) of Arabidopsis acts as a functional KTI when produced in bacteria and in planta. Expression of AtKTI1 is induced late in response to bacterial and fungal elicitors and to salicylic acid. RNAi silencing of the AtKTI1 gene results in enhanced lesion development after infiltration of leaf tissue with the PCD-eliciting fungal toxin fumonisin B1 (FB1) or the avirulent bacterial pathogen Pseudomonas syringae pv tomato DC3000 carrying avrB (Pst avrB). Overexpression of AtKTI1 results in reduced lesion development after Pst avrB and FB1 infiltration. Interestingly, RNAi silencing of AtKTI1 leads to enhanced resistance to the virulent pathogen Erwinia carotovora subsp. carotovora SCC1, while overexpression of AtKTI1 leads to higher susceptibility towards this pathogen. Together, these data indicate that AtKTI1 is involved in modulating PCD in plant–pathogen interactions.

Repression of the Auxin Response Pathway Increases Arabidopsis Susceptibility to Necrotrophic Fungi

2008-05-29

In plants, resistance to necrotrophic pathogens depends on the interplay between different hormone systems, such as those regulated by salicylic acid (SA), jasmonic acid (JA), ethylene, and abscisic acid. Repression of auxin signaling by the SA pathway was recently shown to contribute to antibacterial resistance. Here, we demonstrate that Arabidopsis auxin signaling mutants axr1, axr2, and axr6 that have defects in the auxin-stimulated SCF (Skp1–Cullin–F-box) ubiquitination pathway exhibit increased susceptibility to the necrotrophic fungi Plectosphaerella cucumerina and Botrytis cinerea. Also, stabilization of the auxin transcriptional repressor AXR3 that is normally targeted for removal by the SCF-ubiquitin/proteasome machinery occurs upon P. cucumerina infection. Pharmacological inhibition of auxin transport or proteasome function each compromise necrotroph resistance of wild-type plants to a similar extent as in non-treated auxin response mutants. These results suggest that auxin signaling is important for resistance to the necrotrophic fungi P. cucumerina and B. cinerea. SGT1b (one of two Arabidopsis SGT1 genes encoding HSP90/HSC70 co-chaperones) promotes the functions of SCF E3-ubiquitin ligase complexes in auxin and JA responses and resistance conditioned by certain Resistance (R) genes to biotrophic pathogens. We find that sgt1b mutants are as resistant to P. cucumerina as wild-type plants. Conversely, auxin/SCF signaling mutants are uncompromised in RPP4-triggered resistance to the obligate biotrophic oomycete, Hyaloperonospora parasitica. Thus, the predominant action of SGT1b in R gene-conditioned resistance to oomycetes appears to be at a site other than assisting SCF E3-ubiquitin ligases. However, genetic additivity of sgt1b axr1 double mutants in susceptibility to H. parasitica suggests that SCF-mediated ubiquitination contributes to limiting biotrophic pathogen colonization once plant–pathogen compatibility is established.

A Lesion-Mimic Syntaxin Double Mutant in Arabidopsis Reveals Novel Complexity of Pathogen Defense Signaling

2008-05-29

The lesion-mimic Arabidopsis mutant, syp121 syp122, constitutively expresses the salicylic acid (SA) signaling pathway and has low penetration resistance to powdery mildew fungi. Genetic analyses of the lesion-mimic phenotype have expanded our understanding of programmed cell death (PCD) in plants. Inactivation of SA signaling genes in syp121 syp122 only partially rescues the lesion-mimic phenotype, indicating that additional defenses contribute to the PCD. Whole genome transcriptome analysis confirmed that SA-induced transcripts, as well as numerous other known pathogen-response transcripts, are up-regulated after inactivation of the syntaxin genes. A suppressor mutant analysis of syp121 syp122 revealed that FMO1, ALD1, and PAD4 are important for lesion development. Mutant alleles of EDS1, NDR1, RAR1, and SGT1b also partially rescued the lesion-mimic phenotype, suggesting that mutating syntaxin genes stimulates TIR-NB-LRR and CC-NB-LRR-type resistances. The syntaxin double knockout potentiated a powdery mildew-induced HR-like response. This required functional PAD4 but not functional SA signaling. However, SA signaling potentiated the PAD4-dependent HR-like response. Analyses of quadruple mutants suggest that EDS5 and SID2 confer separate SA-independent signaling functions, and that FMO1 and ALD1 mediate SA-independent signals that are NPR1-dependent. These studies highlight the contribution of multiple pathways to defense and point to the complexity of their interactions.

Altered Disease Development in the eui Mutants and Eui Overexpressors Indicates that Gibberellins Negatively Regulate Rice Basal Disease Resistance

2008-05-29

Gibberellins (GAs) form a group of important plant tetracyclic diterpenoid hormones that are involved in many aspects of plant growth and development. Emerging evidence implicates that GAs also play roles in stress responses. However, the role of GAs in biotic stress is largely unknown. Here, we report that knockout or overexpression of the Elongated uppermost internode (Eui) gene encoding a GA deactivating enzyme compromises or increases, respectively, disease resistance to bacterial blight (Xanthomonas oryzae pv. oyrzae) and rice blast (Magnaporthe oryzae). Exogenous application of GA₃ and the inhibitor of GA synthesis (uniconazol) could increase disease susceptibility and resistance, respectively, to bacterial blight. Similarly, uniconazol restored disease resistance of the eui mutant and GA₃ decreased disease resistance of the Eui overexpressors to bacterial blight. Therefore, the change of resistance attributes to GA levels. In consistency with this, the GA metabolism genes OsGA20ox2 and OsGA2ox1 were down-regulated during pathogen challenge. We also found that PR1a induction was enhanced but the SA level was decreased in the Eui overexpressor, while the JA level was reduced in the eui mutant. Together, our current study indicates that GAs play a negative role in rice basal disease resistance, with EUI as a positive modulator through regulating the level of bioactive GAs.

Rice Gene Network Inferred from Expression Profiling of Plants Overexpressing OsWRKY13, a Positive Regulator of Disease Resistance

Qiu, D., Xiao, J., Xie, W., Liu, H., Li, X., Xiong, L., Wang, S. — 2008-05-29

Accumulating information indicates that plant disease resistance signaling pathways frequently interact with other pathways regulating developmental processes or abiotic stress responses. However, the molecular mechanisms of these types of crosstalk remain poorly understood in most cases. Here we report that OsWRKY13, an activator of rice resistance to both bacterial and fungal pathogens, appears to function as a convergent point for crosstalk among the pathogen-induced salicylate-dependent defense pathway and five other physiologic pathways. Genome-wide analysis of the expression profiles of OsWRKY13-overexpressing lines suggests that OsWRKY13 directly or indirectly regulates the expression of more than 500 genes that are potentially involved in different physiologic processes according to the classification of the Gene Ontology database. By comparing the expression patterns of genes functioning in known pathways or cellular processes of pathogen infection and the phenotypes between OsWRKY13-overexpressing and wild-type plants, our data suggest that OsWRKY13 is also a regulator of other physiologic processes during pathogen infection. The OsWRKY13-associated disease resistance pathway synergistically interacts via OsWRKY13 with the glutathione/glutaredoxin system and flavonoid biosynthesis pathway to monitor redox homeostasis and to putatively enhance the biosynthesis of antimicrobial flavonoid phytoalexins, respectively, in OsWRKY13-overexpressing lines. Meanwhile, the OsWRKY13-associated disease resistance pathway appears to interact antagonistically with the SNAC1-mediated abiotic stress defense pathway, jasmonic acid signaling pathway, and terpenoid metabolism pathway via OsWRKY13 to suppress salt and cold defense responses as well as to putatively retard rice growth and development.

Strong Suppression of Systemic Acquired Resistance in Arabidopsis by NRR is Dependent on its Ability to Interact with NPR1and its Putative Repression Domain

Chern, M., Canlas, P. E., Ronald, P. C. — 2008-05-29

Systemic Acquired Resistance (SAR) in plants confers lasting broad-spectrum resistance to pathogens and requires the phytohormone salicylic acid (SA). Arabidopsis NPR1/NIM1 is a key regulator of the SAR response. Studies attempting to reveal the function of NPR1 and how it mediates SA signaling have led to isolation of two classes of proteins that interact with NPR1: the first class includes rice NRR, Arabidopsis NIMIN1, NIMIN2, and NIMIN3, and tobacco NIMIN2-like proteins; the second class belongs to TGA transcription factors. We have previously shown that overexpression of NRR in rice suppresses both basal and Xa21-mediated resistance. In order to test whether NRR affects SA-induced, NPR1-mediated SAR, we have transformed Arabidopsis with the rice NRR gene and tested its effects on the defense response. Expression of NRR in Arabidopsis results in suppression of PR gene induction by SAR inducer and resistance to pathogens. These phenotypes are even more severe than those of the npr1-1 mutant. The ability of NRR to suppress PR gene induction and disease resistance is correlated with its ability to bind to NPR1 because two point mutations in NRR, which reduce NPR1 binding, fail to suppress NPR1. In contrast, wild-type and a mutant NRR, which still binds to NPR1 strongly, retain the ability to suppress the SAR response. Replacing the C-terminal 79 amino acids of NRR with the VP16 activation domain turns the fusion protein into a transcriptional co-activator. These results indicate that NRR binds to NPR1 in vivo in a protein complex to inhibit transcriptional activation of PR genes and that NRR contains a transcription repression domain for active repression.

Beyond the Receptor

Jones, R. — 2008-02-20

An Update on Abscisic Acid Signaling in Plants and More ...

2008-02-20

The mode of abscisic acid (ABA) action, and its relations to drought adaptive responses in particular, has been a captivating area of plant hormone research for much over a decade. The hormone triggers stomatal closure to limit water loss through transpiration, as well as mobilizes a battery of genes that presumably serve to protect the cells from the ensuing oxidative damage in prolonged stress. The signaling network orchestrating these various responses is, however, highly complex. This review summarizes several significant advances made within the last few years. The biosynthetic pathway of the hormone is now almost completely elucidated, with the latest identification of the ABA4 gene encoding a neoxanthin synthase, which seems essential for de novo ABA biosynthesis during water stress. This leads to the interesting question on how ABA is then delivered to perception sites. In this respect, regulated transport has attracted renewed focus by the unexpected finding of a shoot-to-root translocation of ABA during drought response, and at the cellular level, by the identification of a ß-galactosidase that releases biologically active ABA from inactive ABA-glucose ester. Surprising candidate ABA receptors were also identified in the form of the Flowering Time Control Protein A (FCA) and the Chloroplastic Magnesium Protoporphyrin-IX Chelatase H subunit (CHLH) in chloroplast-nucleus communication, both of which have been shown to bind ABA in vitro. On the other hand, the protein(s) corresponding to the physiologically detectable cell-surface ABA receptor(s) is (are) still not known with certainty. Genetic and physiological studies based on the guard cell have reinforced the central importance of reversible phosphorylation in modulating rapid ABA responses. Sucrose Non-Fermenting Related Kinases (SnRK), Calcium-Dependent Protein Kinases (CDPK), Protein Phosphatases (PP) of the 2C and 2A classes figure as prominent regulators in this single-cell model. Identifying their direct in vivo targets of regulation, which may include H⁺-ATPases, ion channels, 14-3-3 proteins and transcription factors, will logically be the next major challenge. Emerging evidence also implicates ABA as a repressor of innate immune response, as hinted by the highly similar roster of genes elicited by certain pathogens and ABA. Undoubtedly, the most astonishing revelation is that ABA is not restricted to plants and mosses, but overwhelming evidence now indicates that it also exists in metazoans ranging from the most primitive to the most advance on the evolution scale (sponges to humans). In metazoans, ABA has healing properties, and plays protective roles against both environmental and pathogen related injuries. These cross-kingdom comparisons have shed light on the surprising ancient origin of ABA and its attendant mechanisms of signal transduction.

Nitric Oxide in Plants: Production and Cross-talk with Ca2+ Signaling

2008-02-20

Nitric oxide (NO) is a diatomic gas that performs crucial functions in a wide array of physiological processes in animals. The past several years have revealed much about its roles in plants. It is well established that NO is synthesized from nitrite by nitrate reductase (NR) and via chemical pathways. There is increasing evidence for the occurrence of an alternative pathway in which NO production is catalysed from L-arginine by a so far non-identified enzyme. Contradictory results have been reported regarding the respective involvement of these enzymes in specific physiological conditions. Although much remains to be proved, we assume that these inconsistencies can be accounted for by the limited specificity of the pharmacological agents used to suppress NO synthesis but also by the reduced content of L-arginine as well as the inactivity of nitrate-permeable anion channels in nitrate reductase- and/or nitrate/nitrite-deficient plants. Another unresolved issue concerns the molecular mechanisms underlying NO effects in plants. Here, we provide evidence that the second messenger Ca²⁺, as well as protein kinases including MAPK and SnRK2, are very plausible mediators of the NO signals. These findings open new perspectives about NO-based signaling in plants.

Auxin as a Model for the Integration of Hormonal Signal Processing and Transduction

2008-02-20

The regulation of plant growth responds to many stimuli. These responses allow environmental adaptation, thereby increasing fitness. In many cases, the relay of information about a plant's environment is through plant hormones. These messengers integrate environmental information into developmental pathways to determine plant shape. This review will use, as an example, auxin in the root of Arabidopsis thaliana to illustrate the complex nature of hormonal signal processing and transduction. It will then make the case that the application of a systems-biology approach is necessary, if the relationship between a plant's environment and its growth/developmental responses is to be properly understood.

Calcineurin-B-Like Protein CBL9 Interacts with Target Kinase CIPK3 in the Regulation of ABA Response in Seed Germination

Pandey, G. K., Grant, J. J., Cheong, Y. H., Kim, B.-G., Li, L. G., Luan, S. — 2008-02-20

Calcium plays a vital role as a second messenger in many signaling pathways in plants. The calcineurin B-like proteins (CBLs) represent a family of plant calcium-binding proteins that function in calcium signaling by interacting with their interacting protein kinases (CIPKs). In our previous study, we have reported a role for one of the CBLs (CBL9) and one of the CIPKs (CIPK3) in ABA signaling. Here, we have shown that CBL9 and CIPK3 physically and functionally interact with each other in regulating the ABA responses. The CBL9 and CIPK3 proteins interacted with each other in the yeast two-hybrid system and when expressed in plant cells. The double mutant cbl9cipk3 showed the similar hypersensitive response to ABA as observed in single mutants (cbl9 or cipk3). The constitutively active form of CIPK3 genetically complemented the cbl9 mutant, indicating that CIPK3 function downstream of CBL9. Based on these findings, we conclude that CBL9 and CIPK3 act together in the same pathway for regulating ABA responses.

Phosphoinositide and Inositolpolyphosphate Signalling in Defense Responses of Arabidopsis thaliana Challenged by Mechanical Wounding

Mosblech, A., Konig, S., Stenzel, I., Grzeganek, P., Feussner, I., Heilmann, I. — 2008-02-20

Various biochemical signals are implicated in Arabidopsis wound signalling, including jasmonic acid (JA), salicylic acid, auxin, and Ca²⁺. Here, we report on cross-talk of phytohormones with phosphoinositide signals not previously implicated in plant wound responses. Within 30 min of mechanical wounding of Arabidopsis rosette-leaves, the levels of the lipid-derived soluble inositolpolyphosphate, inositol 1,4,5-trisphosphate (InsP₃), increased four to five-fold. Concomitantly, the precursor lipids, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate and phosphatidylinositol transiently depleted, followed by re-synthesis after 30–60 min of stimulation. Increased InsP₃ levels with wounding coincided with JA increases over the first hours of stimulation. In dde2-2-mutant plants deficient in JA biosynthesis, no InsP₃ increase was observed upon wounding, indicating that JA was required for InsP₃ formation, and InsP₃ levels increased in wild-type plants challenged with sorbitol, increasing endogenous JA levels. In InsP 5-ptase plants with attenuated phosphoinositide signalling, the induction of wounding-inducible genes was diminished compared with wild-type plants, suggesting a role for phosphoinositide signalling in mediating plant wound responses. The gene-expression patterns suggest that phosphoinositides contribute to both JA-dependent and JA-independent aspects of wound signalling. Weight gain of Plutella xylostella caterpillars feeding on InsP 5-ptase plants was increased compared with that of caterpillars feeding on wild-type plants. The ecophysiological relevance of phosphoinositide signals in plant defense responses to herbivory is discussed in light of recent findings of inositolpolyphosphate involvement in phytohormone-receptor function.

Dual Functions of Phospholipase D{alpha}1 in Plant Response to Drought

Hong, Y., Zheng, S., Wang, X. — 2008-02-20

Phospholipase D1 (PLD1) has been shown to mediate the abscisic acid regulation of stomatal movements. Arabidopsis plants deficient in PLD1 increased, whereas PLD1-overexpressing tobacco decreased, transpirational water loss. In the early stage of drought, the decrease in water loss was associated with a rapid stomatal closure caused by a high level of PLD in PLD1-overexpressing plants. However, in the late stage of drought, the overexpressing plants displayed more susceptibility to drought than control plants. PLD1 activity in the overexpressing plants was much higher than that of control plants in which drought also induced an increase in PLD1 activity. The high level of PLD1 activity was correlated to membrane degradation in late stages of drought, as demonstrated by ionic leakage and lipid peroxidation. These findings indicate that a high level of PLD1 expression has different effects on plant response to water deficits. It promotes stomatal closure at earlier stages, but disrupts membranes in prolonged drought stress. These findings are discussed in relation to the understanding of PLD functions and potential applications.

Measuring NO Production by Plant Tissues and Suspension Cultured Cells

Vitecek, J., Reinohl, V., Jones, R. L. — 2008-02-20

We describe an inexpensive and reliable detector for measuring NO emitted in the gas phase from plants. The method relies on the use of a strong oxidizer to convert NO to NO₂ and subsequent capture of NO₂ by a Griess reagent trap. The set-up approaches the sensitivity for NO comparable to that of instruments based on chemiluminescence and photoacoustic detectors. We demonstrate the utility of our set-up by measuring NO produced by a variety of well established plant sources. NO produced by nitrate reductase (NR) in tobacco leaves and barley aleurone was readily detected, as was the production of NO from nitrite by the incubation medium of barley aleurone. Arabidopsis mutants that overproduce NO or lack NO-synthase (AtNOS1) also displayed the expected NO synthesis phenotype when assayed by our set-up. We could also measure NO production from elicitor-treated suspension cultured cells using this set-up. Further, we have focused on the detection of NO by a widely used fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM). Our work points to the pitfalls that must be avoided when using DAF-FM to detect the production of NO by plant tissues. In addition to the dramatic effects that pH can have on fluorescence from DAF-FM, the widely used NO scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) can produce anomalous and unexpected results. Perhaps the most serious drawback of DAF-FM is its ability to bind to dead cells and remain NO-sensitive.

Characterization of Gibberellin Receptor Mutants of Barley (Hordeum vulgare L.)

2008-02-20

The sequence of Gid1 (a gene for a gibberellin (GA) receptor from rice) was used to identify a putative orthologue from barley. This was expressed in E. coli, and produced a protein that was able to bind GA in vitro with both structural specificity and saturability. Its potential role in GA responses was investigated using barley mutants with reduced GA sensitivity (gse1 mutants). Sixteen different gse1 mutants each carried a unique nucleotide substitution in this sequence. In all but one case, these changes resulted in single amino acid substitutions, and, for the remaining mutant, a substitution in the 5’ untranslated region of the mRNA is proposed to interfere with translation initiation. There was perfect linkage in segregating populations between new mutant alleles and the gse1 phenotype, leading to the conclusion that the putative GID1 GA receptor sequence in barley corresponds to the Gse1 locus. Determination of endogenous GA contents in one of the mutants revealed enhanced accumulation of bioactive GA₁, and a deficit of C₂₀ GA precursors. All of the gse1 mutants had reduced sensitivity to exogenous GA₃, and to AC94377 (a GA analogue) at concentrations that are normally ‘saturating‘, but, at much higher concentrations, there was often a considerable response. The comparison between barley and rice mutants reveals interesting differences between these two cereal species in GA hormonal physiology.

Selective Deactivation of Gibberellins below the Shoot Apex is Critical to Flowering but Not to Stem Elongation of Lolium

2008-02-20

Gibberellins (GAs) cause dramatic increases in plant height and a genetic block in the synthesis of GA₁ explains the dwarfing of Mendel's pea. For flowering, it is GA₅ which is important in the long-day (LD) responsive grass, Lolium. As we show here, GA₁ and GA₄ are restricted in their effectiveness for flowering because they are deactivated by C-2 hydroxylation below the shoot apex. In contrast, GA₅ is effective because of its structural protection at C-2. Excised vegetative shoot tips rapidly degrade [¹⁴C]GA₁, [¹⁴C]GA₄, and [¹⁴C]GA₂₀ (>80% in 6 h), but not [¹⁴C]GA₅. Coincidentally, genes encoding two 2β-oxidases and a putative 16–17-epoxidase were most expressed just below the shoot apex (<3 mm). Further down the immature stem (>4 mm), expression of these GA deactivation genes is reduced, so allowing GA₁ and GA₄ to promote sub-apical stem elongation. Subsequently, GA degradation declines in florally induced shoot tips and these GAs can become active for floral development. Structural changes which stabilize GA₄ confirm the link between florigenicity and restricted GA 2β-hydroxylation (e.g. 2-hydroxylation and C-2 di-methylation). Additionally, a 2-oxidase inhibitor (Trinexapac Ethyl) enhanced the activity of applied GA₄, as did limiting C-16,17 epoxidation in 16,17-dihydro GAs or after C-13 hydroxylation. Overall, deactivation of GA₁ and GA₄ just below the shoot apex effectively restricts their florigenicity in Lolium and, conversely, with GA₅, C-2 and C-13 protection against deactivation allows its high florigenicity. Speculatively, such differences in GA access to the shoot apex of grasses may be important for separating floral induction from inflorescence emergence and thus could influence their survival under conditions of herbivore predation.

Subcellular Localization and In Vivo Interactions of the Arabidopsis thaliana Ethylene Receptor Family Members

Grefen, C., Stadele, K., Ruzicka, K., Obrdlik, P., Harter, K., Horak, J. — 2008-02-20

The gaseous phytohormone ethylene regulates many developmental processes and responses to environmental conditions in higher plants. In Arabidopsis thaliana, ethylene perception and initiation of signaling are mediated by a family of five receptors which are related to prokaryotic two-component sensor histidine kinases. The transient expression of fluorescence-tagged receptors in tobacco (Nicotiana benthamiana) epidermal leaf cells demonstrated that all ethylene receptors are targeted to the ER endomembrane network and do not localize to the plasmalemma. In support of in planta overlay studies, the ethylene receptors form homomeric and heteromeric protein complexes at the ER in living plant cells, as shown by membrane recruitment assays. A comparable in vivo interaction pattern was found in the yeast mating-based split-ubiquitin system. The overlapping but distinct expression pattern of the ethylene receptor genes suggests a differential composition of the ethylene receptor complexes in different plant tissues. Our findings may have crucial functional implications on the ethylene receptor-mediated efficiency of hormone perception, induction of signaling, signal attenuation and output.

Comprehensive Transcriptome Analysis of Auxin Responses in Arabidopsis

2008-02-20

In plants, the hormone auxin shapes gene expression to regulate growth and development. Despite the detailed characterization of auxin-inducible genes, a comprehensive overview of the temporal and spatial dynamics of auxin-regulated gene expression is lacking. Here, we analyze transcriptome data from many publicly available Arabidopsis profiling experiments and assess tissue-specific gene expression both in response to auxin concentration and exposure time and in relation to other plant growth regulators. Our analysis shows that the primary response to auxin over a wide range of auxin application conditions and in specific tissues comprises almost exclusively the up-regulation of genes and identifies the most robust auxin marker genes. Tissue-specific auxin responses correlate with differential expression of Aux/IAA genes and the subsequent regulation of context- and sequence-specific patterns of gene expression. Changes in transcript levels were consistent with a distinct sequence of conjugation, increased transport capacity and down-regulation of biosynthesis in the temperance of high cellular auxin concentrations. Our data show that auxin regulates genes associated with the biosynthesis, catabolism and signaling pathways of other phytohormones. We present a transcriptional overview of the auxin response. Specific interactions between auxin and other phytohormones are highlighted, particularly the regulation of their metabolism. Our analysis provides a roadmap for auxin-dependent processes that underpins the concept of an ‘auxin code’—a tissue-specific fingerprint of gene expression that initiates specific developmental processes.

Regulation of the Arabidopsis GSK3-like Kinase BRASSINOSTEROID-INSENSITIVE 2 through Proteasome-Mediated Protein Degradation

Peng, P., Yan, Z., Zhu, Y., Li, J. — 2008-02-20

Glycogen synthase kinase 3 (GSK3) is a unique serine/threonine kinase that is implicated in a variety of cellular processes and is regulated by phosphorylation or protein–protein interaction in animal cells. BIN2 is an Arabidopsis GSK3-like kinase that negatively regulates brassinosteroid (BR) signaling. Genetic studies suggested that BIN2 is inhibited in response to BR perception at the cell surface to relieve its inhibitory effects on downstream targets; however, little is known about biochemical mechanisms of its inhibition. Here, we show that BIN2 is regulated by proteasome-mediated protein degradation. Exogenous application of a BR biosynthesis inhibitor and an active BR increased and decreased the amount of BIN2 proteins, respectively. Interestingly, the gain-of-function bin2-1 mutation significantly stabilizes BIN2, making it unresponsive to BR-induced BIN2 depletion. Exogenous application of different plant growth hormones revealed that BIN2 depletion is specifically induced by BR through a functional BR receptor, while treatment of a proteasome inhibitor, MG132, not only prevented the BR-induced BIN2 depletion but also nullified the inhibitory effect of BR on the BIN2 kinase activity. Taken together, our results strongly suggest that proteasome-mediated protein degradation constitutes an important regulatory mechanism for restricting the BIN2 activity.

Functional Interaction of the SNARE Protein NtSyp121 in Ca2+ Channel Gating, Ca2+ Transients and ABA Signalling of Stomatal Guard Cells

Sokolovski, S., Hills, A., Gay, R. A., Blatt, M. R. — 2008-02-20

There is now growing evidence that membrane vesicle trafficking proteins, especially of the superfamily of SNAREs, are critical for cellular signalling in plants. Work from this laboratory first demonstrated that a soluble, inhibitory (dominant-negative) fragment of the SNARE NtSyp121 blocked K⁺ and Cl^– channel responses to the stress-related hormone abscisic acid (ABA), but left open a question about functional impacts on signal intermediates, especially on Ca²⁺-mediated signalling events. Here, we report one mode of action for the SNARE mediated directly through alterations in Ca²⁺ channel gating and its consequent effects on cytosolic-free [Ca²⁺] ([Ca²⁺]_i) elevation. We find that expressing the same inhibitory fragment of NtSyp121 blocks ABA-evoked stomatal closure, but only partially suppresses stomatal closure in the presence of the NO donor, SNAP, which promotes [Ca²⁺]_i elevation independently of the plasma membrane Ca²⁺ channels. Consistent with these observations, Ca²⁺ channel gating at the plasma membrane is altered by the SNARE fragment in a manner effective in reducing the potential for triggering a rise in [Ca²⁺]_i, and we show directly that its expression in vivo leads to a pronounced suppression of evoked [Ca²⁺]_i transients. These observations offer primary evidence for the functional coupling of the SNARE with Ca²⁺ channels at the plant cell plasma membrane and, because [Ca²⁺]_i plays a key role in the control of K⁺ and Cl^– channel currents in guard cells, they underscore an important mechanism for SNARE integration with ion channel regulation during stomatal closure.

The Root Cap Determines Ethylene-Dependent Growth and Development in Maize Roots

Hahn, A., Zimmermann, R., Wanke, D., Harter, K., Edelmann, H. G. — 2008-02-20

Besides providing protection against mechanical damage to the root tip, the root cap is involved in the perception and processing of diverse external and internal stimuli resulting in altered growth and development. The transduction of these stimuli includes hormonal signaling pathways such as those of auxin, ethylene and cytokinin. Here, we show that the root cap is essential for the ethylene-induced regulation of elongation growth and root hair formation in maize. Exogenously applied ethylene is no longer able to inhibit elongation growth when the root cap has been surgically removed prior to hormone treatment. Reconstitution of the cap positively correlates with the developing capacity of the roots to respond to ethylene again. In contrast, the removal of the root cap does not per se affect growth inhibition controlled by auxin and cytokinin. Furthermore, our semi-quantitative RT-PCR results support earlier findings that the maize root cap is a site of high gene expression activity with respect to sensing and responding to hormones such as ethylene. From these data, we propose a novel function of the root cap which is the establishment of competence to respond to ethylene in the distal zones of the root.

Chemical Genetic Dissection of Brassinosteroid-Ethylene Interaction

Gendron, J. M., Haque, A., Gendron, N., Chang, T., Asami, T., Wang, Z.-Y. — 2008-02-20

We undertook a chemical genetics screen to identify chemical inhibitors of brassinosteroid (BR) action. From a chemical library of 10,000 small molecules, one compound was found to inhibit hypocotyl length and activate the expression of a BR-repressed reporter gene (CPD::GUS) in Arabidopsis, and it was named brassinopride (BRP). These effects of BRP could be reversed by co-treatment with brassinolide, suggesting that BRP either directly or indirectly inhibits BR biosynthesis. Interestingly, the compound causes exaggerated apical hooks, similar to that caused by ethylene treatment. The BRP-induced apical hook phenotype can be blocked by a chemical inhibitor of ethylene perception or an ethylene-insensitive mutant, suggesting that, in addition to inhibiting BR, BRP activates ethylene response. Analysis of BRP analogs provided clues about structural features important for its effects on two separate targets in the BR and ethylene pathways. Analyses of the responses of various BR and ethylene mutants to BRP, ethylene, and BR treatments revealed modes of cross-talk between ethylene and BR in dark-grown seedlings. Our results suggest that active downstream BR signaling, but not BR synthesis or a BR gradient, is required for ethylene-induced apical hook formation. The BRP-related compounds can be useful tools for manipulating plant growth and studying hormone interactions.

Ethylene Controls Autophosphorylation of the Histidine Kinase Domain in Ethylene Receptor ETR1

Voet-van-Vormizeele, J., Groth, G. — 2008-02-20

Perception of the phytohormone ethylene is accomplished by a small family of integral membrane receptors. In Arabidopsis, five ethylene receptor proteins are known, including ethylene resistant 1 (ETR1). The hydrophobic amino-terminal domain of these receptors contains the ethylene-binding site while the carboxyl-terminal part consists of a histidine kinase domain and a response regulator domain, which are well known elements found in bacterial two-component signaling. The soluble membrane-extrinsic carboxyl-terminal part of the receptor, which is likely to play an important role in signal transduction, showed intrinsic kinase activity when expressed and purified on its own. However, a correlation between signal input and autokinase activity was not established in these studies, as receptors were missing the transmembrane amino-terminal sensor domain. Thus, it is still unclear whether autophosphorylation occurs in response to perception of the ethylene signal. Here, we report on autophosphorylation studies of purified full-length ETR1. Autokinase activity of the purified receptor is controlled by ethylene or by ethylene agonists like the -acceptor compound cyanide. In fact, both signal molecules were able to completely turn off the intrinsic kinase activity. Furthermore, the observed inhibition of autophosphorylation in ETR1 by both molecules could be prevented when the ethylene antagonist 1-methyl-cyclopropene (MCP) was applied.

Interactions between Axillary Branches of Arabidopsis

Ongaro, V., Bainbridge, K., Williamson, L., Leyser, O. — 2008-02-20

Studies of apical dominance have benefited greatly from two-branch assays in pea and bean, in which the shoot system is trimmed back to leave only two active cotyledonary axillary branches. In these two-branch shoots, a large body of evidence shows that one actively growing branch is able to inhibit the growth of the other, prompting studies on the nature of the inhibitory signals, which are still poorly understood. Here, we describe the establishment of two-branch assays in Arabidopsis, using consecutive branches on the bolting stem. As with the classical studies in pea and bean, these consecutive branches are able to inhibit one another's growth. Not only can the upper branch inhibit the lower branch, but also the lower branch can inhibit the upper branch, illustrating the bi-directional action of the inhibitory signals. Using mutants, we show that the inhibition is partially dependent on the MAX pathway and that while the inhibition is clearly transmitted across the stem from the active to the inhibited branch, the vascular connectivity of the two branches is weak, and the MAX pathway is capable of acting unilaterally in the stem.

A New 'Silk Road' for a New Era of Plant Science

Luan, S., Chen, X.-Y., Raikhel, N., Briggs, W. — 2007-12-21

In the Light of Day: Plant Photomorphogenesis

Briggs, W. R. — 2007-12-21

Chemically Induced and Light-Independent Cryptochrome Photoreceptor Activation

Rosenfeldt, G., Viana, R. M., Mootz, H. D., von Arnim, A. G., Batschauer, A. — 2007-12-21

The cryptochrome photoreceptors of higher plants are dimeric proteins. Their N-terminal photosensory domain mediates dimerization, and the unique C-terminal extension (CCT) mediates signaling. We made use of the human FK506-binding protein (FKBP) that binds with high affinity to rapamycin or rapamycin analogs (rapalogs). The FKBP–rapamycin complex is recognized by another protein, FRB, thus allowing rapamycin-induced dimerization of two target proteins. Here we demonstrate by bioluminescence resonance energy transfer (BRET) assays the applicability of this regulated dimerization system to plants. Furthermore, we show that fusion proteins consisting of the C-terminal domain of Arabidopsis cryptochrome 2 fused to FKBP and FRB and coexpressed in Arabidopsis cells specifically induce the expression of cryptochrome-controlled reporter and endogenous genes in darkness upon incubation with the rapalog. These results demonstrate that the activation of cryptochrome signal transduction can be chemically induced in a dose-dependent fashion and uncoupled from the light signal, and provide the groundwork for gain-of-function experiments to study specifically the role of photoreceptors in darkness or in signaling cross-talk even under light conditions that activate members of all photoreceptor families.

Leaf Positioning of Arabidopsis in Response to Blue Light

Inoue, S.-i., Kinoshita, T., Takemiya, A., Doi, M., Shimazaki, K.-i. — 2007-12-21

Appropriate leaf positioning is essential for optimizing photosynthesis and plant growth. However, it has not been elucidated how green leaves reach and maintain their position for capturing light. We show here the regulation of leaf positioning under blue light stimuli. When 1-week-old Arabidopsis seedlings grown under white light were transferred to red light (25 µmol m^–2 s^–1) for 5 d, new petioles that appeared were almost horizontal and their leaves were curled and slanted downward. However, when a weak blue light from above (0.1 µmol m^–2 s^–1) was superimposed on red light, the new petioles grew obliquely upward and the leaves were flat and horizontal. The leaf positioning required both phototropin1 (phot1) and nonphototropic hypocotyl 3 (NPH3), and resulted in enhanced plant growth. In an nph3 mutant, neither optimal leaf positioning nor leaf flattening by blue light was found, and blue light-induced growth enhancement was drastically reduced. When blue light was increased from 0.1 to 5 µmol m^–2 s^–1, normal leaf positioning and leaf flattening were induced in both phot1 and nph3 mutants, suggesting that phot2 signaling became functional and that the signaling was independent of phot1 and NPH3 in these responses. When plants were irradiated with blue light (0.1 µmol m^–2 s^–1) from the side and red light from above, the new leaves became oriented toward the source of blue light. When we transferred these plants to both blue light and red light from above, the leaf surface changed its orientation to the new blue light source within a few hours, whereas the petioles initially were unchanged but then gradually rotated, suggesting the plasticity of leaf positioning in response to blue light. We showed the tissue expression of NPH3 and its plasma membrane localization via the coiled-coil domain and the C-terminal region. We conclude that NPH3-mediated phototropin signaling optimizes the efficiency of light perception by inducing both optimal leaf positioning and leaf flattening, and enhances plant growth.

The Development of Protein Microarrays and Their Applications in DNA Protein and Protein Protein Interaction Analyses of Arabidopsis Transcription Factors

2007-12-21

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein–DNA and protein–protein interaction analyses. For protein–DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein–DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale.

Role of Arabidopsis RAP2.4 in Regulating Light- and Ethylene-Mediated Developmental Processes and Drought Stress Tolerance

Lin, R.-C., Park, H.-J., Wang, H.-Y. — 2007-12-21

Light and the plant hormone ethylene regulate many aspects of plant growth and development in an overlapping and interdependent fashion. Little is known regarding how their signal transduction pathways cross-talk to regulate plant development in a coordinated manner. Here, we report functional characterization of an AP2/DREB-type transcription factor, Arabidopsis RAP2.4, in mediating light and ethylene signaling. Expression of the RAP2.4 gene is down-regulated by light but up-regulated by salt and drought stresses. RAP2.4 protein is constitutively targeted to the nucleus and it can bind to both the ethylene-responsive GCC-box and the dehydration-responsive element (DRE). We show that RAP2.4 protein possesses an intrinsic transcriptional activation activity in yeast cells and that it can activate a reporter gene driven by the DRE cis-element in Arabidopsis protoplasts. Overexpression of RAP2.4 or mutation in RAP2.4 cause altered expression of representative light-, ethylene-, and drought-responsive genes. Although no salient phenotype was observed with a rap2.4 loss-of-function mutant, constitutive overexpression of RAP2.4 results in defects in multiple developmental processes regulated by light and ethylene, including hypocotyl elongation and gravitropism, apical hook formation and cotyledon expansion, flowering time, root elongation, root hair formation, and drought tolerance. Based on these observations, we propose that RAP2.4 acts at or downstream of a converging point of light and ethylene signaling pathways to coordinately regulate multiple developmental processes and stress responses.

The Clock Protein CCA1 and the bZIP Transcription Factor HY5 Physically Interact to Regulate Gene Expression in Arabidopsis

Andronis, C., Barak, S., Knowles, S. M., Sugano, S., Tobin, E. M. — 2007-12-21

The circadian clock regulates the expression of an array of Arabidopsis genes such as those encoding the LIGHT-HARVESTING CHLOROPHYLL A/B (Lhcb) proteins. We have previously studied the promoters of two of these Arabidopsis genes—Lhcb1*1 and Lhcb1*3—and identified a sequence that binds the clock protein CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). This sequence, designated CCA1-binding site (CBS), is necessary for phytochrome and circadian responsiveness of these genes. In close proximity to this sequence, there exists a G-box core element that has been shown to bind the bZIP transcription factor HY5 in other light-regulated plant promoters. In the present study, we examined the importance of the interaction of transcription factors binding the CBS and the G-box core element in the control of normal circadian rhythmic expression of Lhcb genes. Our results show that HY5 is able to specifically bind the G-box element in the Lhcb promoters and that CCA1 can alter the binding activity of HY5. We further show that CCA1 and HY5 can physically interact and that they can act synergistically on transcription in a yeast reporter gene assay. An absence of HY5 leads to a shorter period of Lhcb1*1 circadian expression but does not affect the circadian expression of CATALASE3 (CAT3), whose promoter lacks a G-box element. Our results suggest that interaction of the HY5 and CCA1 proteins on Lhcb promoters is necessary for normal circadian expression of the Lhcb genes.

Evidence of a Light-Sensing Role for Folate in Arabidopsis Cryptochrome Blue-Light Receptors

Hoang, N., Bouly, J.-P., Ahmad, M. — 2007-12-21

Arabidopsis cryptochromes cry1 and cry2 are blue-light signalling molecules with significant structural similarity to photolyases—a class of blue-light-sensing DNA repair enzymes. Like photolyases, purified plant cryptochromes have been shown to bind both flavin and pterin chromophores. The flavin functions as a light sensor and undergoes reduction in response to blue light that initiates the signalling cascade. However, the role of the pterin in plant cryptochromes has until now been unknown. Here, we show that the action spectrum for light-dependent degradation of cry2 has a significant peak of activity at 380 nm, consistent with absorption by a pterin cofactor. We further show that cry1 protein expressed in living insect cells responds with greater sensitivity to 380 nm light than to 450 nm, consistent with a light-harvesting antenna pigment that transfers excitation energy to the oxidized flavin of cry1. The pterin biosynthesis inhibitor DHAP selectively reduces cryptochrome responsivity at 380 nm but not 450 nm blue light in these cell cultures, indicating that the antenna pigment is a folate cofactor similar to that of photolyases.

Abscisic Acid, High-Light, and Oxidative Stress Down-Regulate a Photosynthetic Gene via a Promoter Motif Not Involved in Phytochrome-Mediated Transcriptional Regulation

Staneloni, R. J., Rodriguez-Batiller, M. J., Casal, J. J. — 2007-12-21

In etiolated seedlings, light perceived by phytochrome promotes the expression of light-harvesting chlorophyll a/b protein of photosystem II (Lhcb) genes. However, excess of photosynthetically active radiation can reduce Lhcb expression. Here, we investigate the convergence and divergence of phytochrome, high-light stress and abscisic acid (ABA) signaling, which could connect these processes. Etiolated Arabidopsis thaliana seedlings bearing an Lhcb promoter fused to a reporter were exposed to continuous far-red light to activate phytochrome and not photosynthesis, and treated with ABA. We identified a cis-acting region of the promoter required for down-regulation by ABA. This region contains a CCAC sequence recently found to be necessary for ABI4-binding to an Lhcb promoter. However, we did not find a G-box-binding core motif often associated with the ABI4-binding site in genes promoted by light and repressed by ABI4. Mutations involving this motif also impaired the responses to reduced water potential, the response to high photosynthetic light and the response to methyl viologen but not the response to low temperature or to Norflurazon. We propose a model based on current and previous findings, in which hydrogen peroxide produced in the chloroplasts under high light conditions interacts with the ABA signaling network to regulate Lhcb expression. Since the mutation that affects high-light and methyl viologen responses does not affect phytochrome-mediated responses, the regulation by retrograde and phytochrome signaling can finally be separated at the target promoter level.

A Rice Phytochrome A in Arabidopsis: The Role of the N-terminus under red and far-red light

Kneissl, J., Shinomura, T., Furuya, M., Bolle, C. — 2007-12-21

The phytochrome (phy)A and phyB photoreceptors mediate three photobiological response modes in plants; whereas phyA can mediate the very-low-fluence response (VLFR), the high-irradiance response (HIR) and, to some extent, the low fluence response (LFR), phyB and other type II phytochromes only mediate the LFR. To investigate to what level a rice phyA can complement for Arabidopsis phyA or phyB function and to evaluate the role of the serine residues in the first 20 amino acids of the N-terminus of phyA, we examined VLFR, LFR, and HIR responses in phyB and phyAphyB mutant plants transformed with rice PHYA cDNA or a mutant rice PHYA cDNA in which the first 10 serine residues were mutated to alanines (phyA SA). Utilizing mutants without endogenous phyB allowed the evaluation of red-light-derived responses sensed by the rice phyA. In summary, the WT rice phyA could complement VLFR and LFR responses such as inhibition of hypocotyl elongation under pulses of FR or continuous R light, induction of flowering and leaf expansion, whereas the phyA SA was more specific for HIR responses (e.g. inhibition of hypocotyl elongation and anthocyanin accumulation under continuous far-red light). As the N-terminal serines can no longer be phosphorylated in the phyA SA mutant, this suggests a role for phosphorylation discriminating between the different phyA-dependent responses. The efficacy of the rice phyA expressed in Arabidopsis was dependent upon the developmental age of the plants analyzed and on the physiological response, suggesting a stage-dependent downstream modulation of phytochrome signaling.

The Subcellular Localization and Blue-Light-Induced Movement of Phototropin 1-GFP in Etiolated Seedlings of Arabidopsis thalianaw

Wan, Y.-L., Eisinger, W., Ehrhardt, D., Kubitscheck, U., Baluska, F., Briggs, W. — 2007-12-21

Phototropin 1 (phot1) is a photoreceptor for phototropism, chloroplast movement, stomatal opening, leaf expansion, and solar tracking in response to blue light. Following earlier work with PHOT1::GFP (Sakamoto and Briggs, 2002), we investigated the pattern of cellular and subcellular localization of phot1 in 3–4 d old etiolated seedlings of Arabidopsis thalinana. As expressed from native upstream sequences, the PHOT1::GFP fusion protein is expressed strongly in the abaxial tissues of the cotyledons and in the elongating regions of the hypocotyl. It is moderately expressed in the shoot/root transition zone and in cells near the root apex. A fluorescence signal is undetectable in the root epidermis, root cap, and root apical meristem itself. The plasma membranes of mesophyll cells near the cotyledon margin appear labeled uniformly but cross-walls created by recent cell divisions are more strongly labeled. The pattern of labeling of individual cell types varies with cell type and developmental stage. Blue-light treatment causes PHOT1::GFP, initially relatively evenly distributed at the plasma membrane, to become reorganized into a distinct mosaic with strongly labeled punctate areas and other areas completely devoid of fluorescence—a phenomenon best observed in cortical cells in the hypocotyl elongation region. Concomitant with or following this reorganization, PHOT1::GFP moves into the cytoplasm in all cell types investigated except for guard cells. It disappears from the cytoplasm by an unidentified mechanism after several hours in darkness. Neither its appearance in the cytoplasm nor its eventual disappearance in darkness is prevented by the translation inhibitor cycloheximide, although the latter process is retarded. We hypothesize that blue-light-induced phot1 re-localization modulates blue-light-activated signal transduction.

Interaction of the Arabidopsis UV-B-Specific Signaling Component UVR8 with Chromatin

Cloix, C., Jenkins, G. I. — 2007-12-21

Arabidopsis UV RESISTANCE LOCUS8 (UVR8) is a UV-B-specific signaling component that regulates expression of a range of genes concerned with UV protection. Here, we investigate the interaction of UVR8 with chromatin. Using antibodies specific to UVR8 in chromatin immunoprecipitation (ChIP) assays with wild-type plants, we show that native UVR8 binds to chromatin in vivo. Similar experiments using an anti-GFP antibody with plants expressing a GFP–UVR8 fusion show that UVR8 associates with a relatively small region of chromatin containing the HY5 gene. UVR8 interacts with chromatin containing the promoter regions of other genes, but not with all the genes it regulates. UV-B is not required for the interaction of UVR8 with chromatin because association with several gene loci is observed in the absence of UV-B. Pull-down assays demonstrate that UVR8 associates with histones in vivo and competition experiments indicate that the interaction is preferentially with histone H2B. ChIP experiments using antibodies that recognize specific histone modifications indicate that the UV-B-stimulated transcription of some genes may be correlated with histone modification. In particular, the ELIP1 promoter showed a significant enrichment of diacetyl histone H3 (K9/K14) following UV-B exposure. These findings increase understanding of the interaction of the key UV-B-specific regulator UVR8 with chromatin.

Disruptions in AUX1-Dependent Auxin Influx Alter Hypocotyl Phototropism in Arabidopsis

2007-12-21

Phototropism represents a differential growth response by which plant organs can respond adaptively to changes in the direction of incident light to optimize leaf/stem positioning for photosynthetic light capture and root growth orientation for water/nutrient acquisition. Studies over the past few years have identified a number of components in the signaling pathway(s) leading to development of phototropic curvatures in hypocotyls. These include the phototropin photoreceptors (phot1 and phot2) that perceive directional blue-light (BL) cues and then stimulate signaling, leading to relocalization of the plant hormone auxin, as well as the auxin response factor NPH4/ARF7 that responds to changes in local auxin concentrations to directly mediate expression of genes likely encoding proteins necessary for development of phototropic curvatures. While null mutations in NPH4/ARF7 condition an aphototropic response to unidirectional BL, seedlings carrying the same mutations recover BL-dependent phototropic responsiveness if co-irradiated with red light (RL) or pre-treated with either ethylene. In the present study, we identify second-site enhancer mutations in the nph4 background that abrogate these recovery responses. One of these mutations—map1 (modifier of arf7 phenotypes 1)—was found to represent a missense allele of AUX1—a gene encoding a high-affinity auxin influx carrier previously associated with a number of root responses. Pharmocological studies and analyses of additional aux1 mutants confirmed that AUX1 functions as a modulator of hypocotyl phototropism. Moreover, we have found that the strength of dependence of hypocotyl phototropism on AUX1-mediated auxin influx is directly related to the auxin responsiveness of the seedling in question.

Analysis of Natural Allelic Variation Controlling Arabidopsis thaliana Seed Germinability in Response to Cold and Dark: Identification of Three Major Quantitative Trait Loci

Meng, P.-H., Macquet, A., Loudet, O., Marion-Poll, A., North, H. M. — 2007-12-21

Light and temperature are key external factors in the control of Arabidopsis thaliana seed germination and dormancy mechanisms. Perception and response to these stimuli have to ensure that seedling emergence and growth occur at the most advantageous time for correct establishment. Analysis of over 300 Arabidopsis accessions identified 14, from 12 different geographical locations, that were able to germinate to greater than 20% at 6°C in the dark. This natural variation was exploited to identify genetic loci responsible for cold-tolerant, dark germination. A quantitative trait loci approach was used on recombinant inbred line progeny of a cross between Bay-0 and Shahdara. Six distinct quantitative trait loci were identified, three of which were major loci, each responsible for 17–25% of the phenotypic variability in this trait. Parental phenotypes indicated that the majority of the cold-tolerant, dark-germination characteristics are related to light responses. Validation of the three major loci using heterogeneous inbred families confirmed the feasibility of fine mapping and cloning the genes at the quantitative trait loci responsible for cold-tolerant, dark germination.

A Novel Two Domain-Fusion Protein in Cyanobacteria with Similarity to the CAB/ELIP/HLIP Superfamily: Evolutionary Implications and Regulation

Kilian, O., Steunou, A. S., Grossman, A. R., Bhaya, D. — 2007-12-21

Vascular plants contain abundant, light-harvesting complexes in the thylakoid membrane that are non-covalently associated with chlorophylls and carotenoids. These light-harvesting chlorophyll a/b binding (LHC) proteins are members of an extended CAB/ELIP/HLIP superfamily of distantly related polypeptides, which have between one and four transmembrane helices (TMH). This superfamily includes the single TMH, high-light-inducible proteins (Hlips), found in cyanobacteria that are induced by various stress conditions, including high light, and are considered ancestral to the LHC proteins. The roles of, and evolutionary relationships between, these superfamily members are of particular interest, since they function in both light harvesting and photoprotection and may have evolved through tandem gene duplication and fusion events. We have investigated the Hlips (hli gene family) in the thermophilic unicellular cyanobacterium Synechococcus OS-B’. The five hli genes present on the genome of Synechococcus OS-B’ are relatively similar, but transcript analyses indicate that there are different patterns of transcript accumulation when the cells are exposed to various growth conditions, suggesting that different Hlips may have specific functions. Hlip5 has an additional TMH at the N-terminus as a result of a novel fusion event. This additional TMH is very similar to a conserved hypothetical, single membrane-spanning polypeptide present in most cyanobacteria. The evolutionary significance of these results is discussed.

Blue-Light-Independent Activity of Arabidopsis Cryptochromes in the Regulation of Steady-State Levels of Protein and mRNA Expression

2007-12-21

Cryptochromes are blue-light receptors that mediate blue-light inhibition of hypocotyl elongation and blue-light stimulation of floral initiation in Arabidopsis. In addition to their blue-light-dependent functions, cryptochromes are also involved in blue-light-independent regulation of the circadian clock, cotyledon unfolding, and hypocotyl inhibition. However, the molecular mechanism associated with the blue-light-independent function of cryptochromes remains unclear. We reported here a comparative proteomics study of the light regulation of protein expression. We showed that, as expected, the protein expression of many metabolic enzymes changed in response to both blue light and red light. Surprisingly, some light-regulated protein expression changes are impaired in the cry1cry2 mutant in both blue light and red light. This result suggests that, in addition to mediating blue-light-dependent regulation of protein expression, cryptochromes are also involved in the blue-light-independent regulation of gene expression. Consistent with this hypothesis, the cry1cry2 mutant exhibited reduced changes of mRNA expression in response to not only blue light, but also red light, although the cryptochrome effects on the red-light-dependent gene expression changes are generally less pronounced. These results support a hypothesis that, in addition to their blue-light-specific functions, cryptochromes also play roles in the control of gene expression mediated by the red/far-red-light receptor phytochromes.

In Vivo Phosphorylation Site Mapping and Functional Characterization of Arabidopsis Phototropin 1

Sullivan, S., Thomson, C. E., Lamont, D. J., Jones, M. A., Christie, J. M. — 2007-12-21

Phototropins (phot1 and phot2) are blue-light receptor kinases controlling a range of responses that optimize the photosynthetic efficiency of plants. Light sensing is mediated by two flavin-binding motifs, known as LOV1 and LOV2, located within the N-terminal region of the protein. Photoexcitation via LOV2 leads to activation of the C-terminal kinase domain and consequently receptor autophosphorylation. However, knowledge of the in-vivo phosphorylation sites for Arabidopsis phototropins is lacking and has impeded progress in elucidating the functional significance of receptor phosphorylation. We have purified phot1 from Arabidopsis and identified the in-vivo sites of receptor phosphorylation by liquid chromatography tandem mass spectrometry. Arabidopsis-derived phot1 binds flavin mononucleotide as chromophore and is phosphorylated at four major sites located upstream of LOV2 (Ser⁵⁸, Ser⁸⁵, Ser³⁵⁰, and Ser⁴¹⁰), three of which are induced by blue light. Nevertheless, structure-function analysis indicates that the biological activity of phot1 can be attributed to a modular unit comprising the LOV2-kinase region of the protein. Thus, peptide regions upstream of LOV2, including the sites of receptor phosphorylation identified here, do not appear to be important for receptor signaling. By contrast, these regions may be necessary for maximizing stomatal performance and possibly light-induced relocalization of phot1.