Bob B. Buchanan

A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells

The plant hormone abscisic acid (ABA) serves as a physiological monitor to assess the water status of plants and, under drought conditions, induces stomatal pore closure by activating specific ion channels, such as a slow-anion channel (SLAC1) that, in turn, mediate ion efflux from the guard cells. Earlier genetic analyses uncovered a protein kinase (OST1) and several 2C-type phosphatases, as respective positive and negative regulators of ABA-induced stomatal closure. Here we show that the OST1 kinase interacts with the SLAC1 anion channel, leading to its activation via phosphorylation. PP2CA, one of the PP2C phosphatase family members acts in an opposing manner and inhibits the activity of SLAC1 by two mechanisms: (1) direct interaction with SLAC1 itself, and (2) physical interaction with OSTI leading to inhibition of the kinase independently of phosphatase activity. The results suggest that ABA signaling is mediated by a physical interaction chain consisting of several components, including a PP2C member, SnRK2-type kinase (OST1), and an ion channel, SLAC1, to regulate stomatal movements. The findings are in keeping with a paradigm in which a protein kinase-phosphatase pair interacts physically with a target protein to couple a signal with a specific response.

Proteomics gives insight into the regulatory function of chloroplast thioredoxins

Thioredoxins are small multifunctional redox active proteins widely if not universally distributed among living organisms. In chloroplasts, two types of thioredoxins (f and m) coexist and play central roles in regulating enzyme activity. Reduction of thioredoxins in chloroplasts is catalyzed by an iron-sulfur disulfide enzyme, ferredoxin-thioredoxin reductase, that receives photosynthetic electrons from ferredoxin, thereby providing a link between light and enzyme activity. Chloroplast thioredoxins function in the regulation of the Calvin cycle and associated processes. However, the relatively small number of known thioredoxin-linked proteins (about 16) raised the possibility that others remain to be identified. To pursue this opportunity, we have mutated thioredoxins f and m, such that the buried cysteine of the active disulfide has been replaced by serine or alanine, and bound them to affinity columns to trap target proteins of chloroplast stroma. The covalently linked proteins were eluted with DTT, separated on gels, and identified by mass spectrometry. This approach led to the identification of 15 potential targets that function in 10 chloroplast processes not known to be thioredoxin linked. Included are proteins that seem to function in plastid-to-nucleus signaling and in a previously unrecognized type of oxidative regulation. Approximately two-thirds of these targets contained conserved cysteines. We also identified 11 previously unknown and 9 confirmed target proteins that are members of pathways known to be regulated by thioredoxin. In contrast to results with individual enzyme assays, specificity for thioredoxin f or m was not observed on affinity chromatography.

Regulation of CO2 assimilation in oxygenic photosynthesis: The ferredoxin/thioredoxin system: Perspective on its discovery, present status, and future development

Abstract It is well established that light functions in photosynthesis by providing the assimilatory power (ATP and NADPH) for the “dark” reactions, historically known as the synthesis of carbohydrate from carbon dioxide. Research during the past two and a half decades has revealed that light makes yet another contribution to the overall photosynthetic process: to provide specific regulatory agents that enable the dark reactions to function more effectively (Fig. 1). The purpose of this article is to give a brief account of this regulatory function, emphasizing the ferredoxin/thioredoxin system. The discussion includes a brief description of the history of the system, its current status, and how knowledge of the system may be used in the future.

Thioredoxins and Glutaredoxins: Unifying Elements in Redox Biology

Since their discovery as a substrate for ribonucleotide reductase (RNR), the role of thioredoxin (Trx) and glutaredoxin (Grx) has been largely extended through their regulatory function. Both proteins act by changing the structure and activity of a broad spectrum of target proteins, typically by modifying redox status. Trx and Grx are members of families with multiple and partially redundant genes. The number of genes clearly increased with the appearance of multicellular organisms, in part because of new types of Trx and Grx with orthologs throughout the animal and plant kingdoms. The function of Trx and Grx also broadened as cells achieved increased complexity, especially in the regulation arena. In view of these progressive changes, the ubiquitous distribution of Trx and the wide occurrence of Grx enable these proteins to serve as indicators of the evolutionary history of redox regulation. In so doing, they add a unifying element that links the diverse forms of life to one another in an uninterrupted continuum. It is anticipated that future research will embellish this continuum and further elucidate the properties of these proteins and their impact on biology. The new information will be important not only to our understanding of the role of Trx and Grx in fundamental cell processes but also to future societal benefits as the proteins find new applications in a range of fields.

A protein phosphorylation/dephosphorylation network regulates a plant potassium channel.

Potassium (K+) is an essential nutrient for plant growth and development. Plants often adapt to low K+ conditions by increasing their K+ uptake capability. Recent studies have led to the identification of a calcium signaling pathway that enables plants to act in this capacity. Calcium is linked to two calcineurin B-like calcium sensors (CBLs) and a target kinase (CBL-interacting protein kinase 23 or CIPK23) that, in turn, appears to phosphorylate and activate the potassium channel, Arabidopsis K+ transporter 1 (AKT1), responsible for K+ uptake in roots. Here, we report evidence that this regulatory mechanism is more elaborate than earlier envisaged. The recently described pathway is part of an extensive network whereby several CBLs interact with multiple CIPKs in the activation of the potassium channel, AKT1. The physical interactions among the CBL, CIPK, and AKT1 components provide a mechanism for specifying the members of the CBL and CIPK families functional in AKT1 regulation. The interaction between the CIPKs and AKT1 was found to involve the kinase domain of the CIPK component and the ankyrin repeat domain of the channel. Furthermore, we identified a 2C-type protein phosphatase that physically interacts and inactivates the AKT1 channel. These findings provide evidence that the calcium-sensitive CBL and CIPK families together with 2C-type protein phosphatases form a protein phoshporylation/dephosphorylation network that regulates the AKT1 channel for K+ transport in plants.

Thioredoxin targets in plants: The first 30 years

The turn of the century welcomed major developments in redox biology. In plants, proteomics made possible the identification of proteins linked to thioredoxin (Trx), initially in chloroplasts and then other cell compartments. Two procedures, one based on thiol specific probes and the other on mutant Trx proteins, facilitated the labeling or isolation of potential Trx targets that were later identified with proteomic approaches. As a result, the number of targets in land plants increased 10-fold from fewer than 40 to more than 400. Additional targets have been identified in green algae and cyanobacteria, making a grand total of 500 in oxygenic photosynthetic organisms. Collectively these proteins have the potential to influence virtually every major process of the cell. A number of laboratories currently seek to confirm newly identified Trx targets by biochemical and genetic approaches. Almost certainly many new targets become redox active during oxidative stress, enabling the plant to cope with changing environments. Under these conditions, certain targets may be glutathionylated or nitrosylated such that reversion to the original reduced state is facilitated not only by Trx, but also, in some cases preferably, by glutaredoxin. When judging changes linked to Trx, it is prudent to recognize that effects transcend classical light/dark or oxidative regulation and fall in other arenas, in some cases yet to be defined. While future work will continue to give insight into functional details, it is clear that Trx plays a fundamental role in regulating diverse processes of the living cell.

A strategy for the identification of proteins targeted by thioredoxin

Thioredoxins are 12-kDa proteins functional in the regulation of cellular processes throughout the animal, plant, and microbial kingdoms. Growing evidence with seeds suggests that an h-type of thioredoxin, reduced by NADPH via NADP-thioredoxin reductase, reduces disulfide bonds of target proteins and thereby acts as a wakeup call in germination. A better understanding of the role of thioredoxin in seeds as well as other systems could be achieved if more were known about the target proteins. To this end, we have devised a strategy for the comprehensive identification of proteins targeted by thioredoxin. Tissue extracts incubated with reduced thioredoxin are treated with a fluorescent probe (monobromobimane) to label sulfhydryl groups. The newly labeled proteins are isolated by conventional two-dimensional electrophoresis: (i) nonreducing/reducing or (ii) isoelectric focusing/reducing SDS/PAGE. The isolated proteins are identified by amino acid sequencing. Each electrophoresis system offers an advantage: the first method reveals the specificity of thioredoxin in the reduction of intramolecular vs. intermolecular disulfide bonds, whereas the second method improves the separation of the labeled proteins. By application of both methods to peanut seed extracts, we isolated at least 20 thioredoxin targets and identified 5—three allergens (Ara h2, Ara h3, and Ara h6) and two proteins not known to occur in peanut (desiccation-related and seed maturation protein). These findings open the door to the identification of proteins targeted by thioredoxin in a wide range of systems, thereby enhancing our understanding of its function and extending its technological and medical applications.

NTRC links built-in thioredoxin to light and sucrose in regulating starch synthesis in chloroplasts and amyloplasts

Plants have an unusual plastid-localized NADP-thioredoxin reductase C (NTRC) containing both an NADP-thioredoxin reductase (NTR) and a thioredoxin (Trx) domain in a single polypeptide. Although NTRC is known to supply reductant for detoxifying hydrogen peroxide in the dark, its other functions are unknown. We now report that NTRC plays a previously unrecognized role in the redox regulation of ADP-glucose pyrophosphorylase (AGPase), a central enzyme of starch synthesis. When supplied NADPH, NTRC activated AGPase in vitro in a redox reaction that required the active site cysteines of both domains of the enzyme. In leaves, AGPase was activated in planta either by light or external feeding of sucrose in the dark. Leaves of an Arabidopsis NTRC KO mutant showed a decrease both in the extent of redox activation of AGPase and in the enhancement of starch synthesis either in the light (by 40–60%) or in the dark after treatment with external sucrose (by almost 100%). The light-dependent activation of AGPase in isolated chloroplasts, by contrast, was unaffected. In nonphotosynthetic tissue (roots), KO of NTRC decreased redox activation of AGPase and starch synthesis in response to light or external sucrose by almost 90%. The results provide biochemical and genetic evidence for a role of NTRC in regulating starch synthesis in response to either light or sucrose. The data also suggest that the Trx domain of NTRC and, to a lesser extent, free Trxs linked to ferredoxin enable amyloplasts of distant sink tissues to sense light used in photosynthesis by leaf chloroplasts and adjust heterotrophic starch synthesis accordingly.

Severe sensitivity to ultraviolet radiation in an Arabidopsis mutant deficient in flavonoid accumulation

A mutantArabidopsis thaliana L., which displays a dramatic increase in sensitivity to ultraviolet-B (UV-B) radiation compared with wild-type plants, has been isolated by chemical mutagenesis. This mutation appears to affect UV-tolerance specifically, since mutant plants are indistinguishable from wild type with respect to their degree of resistance to other forms of stress. The UV-sensitive mutation proved to be recessive and to segregate as a single Mendelian locus. This single gene defect was shown to lead to a block in the synthesis of a group of flavonoids which normally accumulate in developing wild-typeArabidopsis and which increase in concentration when plants are exposed to UV radiation. One of these compounds has been identified as a rhamnosylated derivative of the flavonol, kaempferol. The results suggest that one or more of the flavonoids whose production has been blocked in the UV-sensitive mutant is essential for the protection of Arabidopsis against UV-radiation damage. This constitutes further evidence that flavonoids play an important role in the protection of plants from the damaging effects of UV-B light.

Mechanisms of CO2 fixation in bacterial photosynthesis studied by the carbon isotope fractionation technique.

Abstract1. The carbon isotope discrimination properties of a representative of each of the three types of photosynthetic bacteria Chlorobium thiosulfatophilum, Rhodospirillum rubrum and Chromatium and of the C3-alga Chlamydomonas reinhardii were determined by measuring the ratio of 13CO2 to 12CO2 incorporated during photoautotrophic growth. 2. Chromatium and R. rubrum had isotope selection properties similar to those of C3-plants, whereas Chlorobium was significantly different. 3. The results suggest that Chromatium and R. rubrum assimilate CO2 mainly via ribulose 1,5-diphosphate carboxylase and the associated reactions of the reductive pentose phosphate cycle, whereas Chlorobium utilizes other mechanisms. Such mechanisms would include the ferredoxin-linked carboxylation enzymes and associated reactions of the reductive carboxylic acid cycle.