Molecular Plant - current issue

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.