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Type-II metacaspases are conserved cysteine proteases found in eukaryotes with oxygenic photosynthesis, including green plants and some algae, such as Chlamydomonas and Volvox. Genetic and biochemical studies showed that some members in this protease family could be involved in oxidative stress-induced cell death in higher plants, but their regulatory mechanisms remain unclear. Biochemically, two distinct classes of type-II metacaspases are exemplified by AtMC4 and AtMC9 from Arabidopsis, with AtMC4 activation dependent on calcium under neutral pH, whereas AtMC9 is active only under mildly acidic pH, regardless of the availability of calcium. Here, we constructed all six possible combinations between the p20, linker, and p10 domains from AtMC4 and AtMC9. Our results show that calcium stimulation of AtMC4 activity is associated with essential amino acids located in its p20 domain. In contrast, the acidic pH optimum trait is lost from AtMC9 if one or two of its domains are replaced by that from AtMC4, suggesting that multiple interactions between domains in AtMC9 may be responsible for this property. Consistent with this, we found conserved 'signature' residues in each of the three domains that distinguish AtMC4- and AtMC9-like classes of metacaspases. Tracing the origin of the AtMC9 class, we found evidence for its appearance between lycophytes and gymnosperms, coincident with the evolution of more complex root archetypes in terrestrial plants. Our work suggests that the distinctive properties of the AtMC9-like protease could be associated with special cellular physiology in the roots of gymnosperms and angiosperms that are distinct from lycophytes., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Key Message: Leveraging the heightened levels of polymorphism in NB-ARC-related protein encoding genes in higher plants, a bioinformatic pipeline was created to identify regions in this gene family from sequenced plant genomes that exhibit fragment length or single nucleotide differences in different accessions of the same species. Testing this approach with the aquatic plant Spirodela polyrhiza demonstrated its superior performance in comparison with currently available genotyping technologies based on PCR amplification. Rapid and economical genotyping tools that can reliably distinguish species and intraspecific variations in plants can be powerful tools for biogeographical and ecological studies. Clones of the cosmopolitan duckweed species, Spirodela polyrhiza, are difficult to distinguish morphologically due to their highly abbreviated architecture and inherently low levels of sequence variation. The use of plastidic markers and generic Amplification Fragment Length Polymorphism approaches have met with limited success in resolving clones of S. polyrhiza from diverse geographical locales. Using whole genome sequencing data from nine S. polyrhiza clones as a training set, we created an informatic pipeline to identify and rank polymorphic regions from nuclear-encoded NB-ARC-related genes to design markers for PCR, Sanger sequencing (barcoding), and fragment length analysis. With seven primer sets, we found 21 unique fingerprints from a set of 23 S. polyrhiza clones. However, three of these clones share the same fingerprint and are indistinguishable by these markers. These primer sets can also be used as interspecific barcoding tools to rapidly resolve S. polyrhiza from the closely related S. intermedia species without the need for DNA sequencing. Our work demonstrates a general approach of using hyper-polymorphic loci within genomes as a resource to produce facile tools that can have high resolving power for genotyping applications.

Metacaspases are cysteine-dependent proteases found in protozoa, fungi and plants and are distantly related to metazoan caspases. Although metacaspases share structural properties with those of caspases, they lack Asp specificity and cleave their targets after Arg or Lys residues. Studies performed over the past 10 years have demonstrated that metacaspases are multifunctional proteases essential for normal physiology of non-metazoan organisms. This article provides a comprehensive overview of the metacaspase function and molecular regulation during programmed cell death, stress and cell proliferation, as well as an analysis of the first metacaspase-mediated proteolytic pathway. To prevent further misapplication of caspase-specific molecular probes for measuring and inhibiting metacaspase activity, we provide a list of probes suitable for metacaspases.

In Nature plants are constantly challenged by a variety of environmental stresses that could lead to disruptions in cellular homeostasis. Programmed cell death (PCD) is a fundamental cellular process that is often associated with defense responses to pathogens, during development and in response to abiotic stresses in fungi, animals and plants. Although there are many characteristics shared between different types of PCD events, it remains unknown whether a common mechanism drives various types of PCD in eukaryotes. One candidate regulator for such a mechanism is Bax Inhibitor-1 (BI-1), an evolutionary conserved, endoplasmic reticulum (ER)-resident protein that represents an ancient cell death regulator that potentially regulates PCD in all eukaryotes. Recent findings strongly suggested that BI-1 plays an important role in the conserved ER stress response pathway to modulate cell death induction in response to multiple types of cell death signals. As ER stress signaling pathways has been suggested to play important roles not only in the control of ER homeostasis but also in other biological processes such as the response to pathogens and abiotic stress in plants, BI-1 might function to control the convergence point that modulates the level of the "pro-survival and pro-death" signals under multiple stress conditions.

The molecular identity of the key executioners involved in controlling plant programmed cell death (PCD) has been elusive. In a recent paper published in Science, Hatsugai and coworkers reported that a well-characterized protease called VPE from the plant cell vacuole can cleave caspase-specific substrates and is required for cell death activation by tobacco mosaic virus. This work provides clear evidence for the importance of the vacuole in plant PCD and a novel regulatory function for this organelle as well as for VPE proteases.

Transcription factors often belong to multigene families and their individual contribution in a particular regulatory network remains difficult to assess. We show here that specific members from a family of conserved Arabidopsis bZIP transcription factors, the TGA proteins, are regulated in their protein stability by developmental stage-specific proteolysis. Using GFP fusions of three different Arabidopsis TGA factors that represent members of distinct subclasses of the TGA factor family, we demonstrate that two of these TGA proteins are specifically targeted for proteolysis in mature leaf cells. Using a supershift gel mobility assay, we found evidence for similar regulation of the cognate proteins as compared to the GFP fusion proteins expressed under the cauliflower mosaic virus (CaMV) 35S promoter. Using various inhibitors, we showed that the expression of at least one of these three TGA factors could be stabilized by inhibition of proteasome-mediated proteolysis. This study indicates that TGA transcription factors may be regulated by distinct pathways of targeted proteolysis that can serve to modulate the contribution of specific members of a multigene family in complex regulatory pathways.

Salicylic acid (SA) is a key regulator for the induction of systemic acquired resistance (SAR), and NPR1 is a critical mediator for the biological effects of SA. Physical interactions between NPR1 and TGA factors, a conserved family of basic-leucine-zipper (bZip) proteins in plants, have suggested a role for these transcription factors in mediating SAR induction via the regulation of defense genes. To elucidate this function, we constructed a trans-dominant mutant that specifically eliminates DNA-binding activities of this class of bZip proteins in transgenic tobacco plants. Our results demonstrate that the loss of TGA DNA-binding activities is correlated with suppression of two xenobiotic-responsive genes, GNT35 and STR246, and enhanced induction of pathogenesis-related (PR) genes by SA. In addition, these TGA-suppressed plants exhibited higher levels of PR gene induction by pathogen challenge and an enhanced SAR. These results suggest that TGA transcription factors serve both negative and positive regulatory roles in mediating plant defense responses.

NPR1 is a critical component of the salicylic acid (SA)-mediated signal transduction pathway leading to the induction of defense genes, such as the pathogenesis-related (PR)-1 gene, and enhanced disease resistance. Using a yeast two-hybrid screen, we identified several NPR1-interacting proteins (NIPs). Two of these NIPs are members of the TGA/OBF family of basic leucine zipper (bZIP) transcription factors; this family has been implicated in the activation of SA-responsive genes, including PR-1. Six TGA family members were tested and shown to differentially interact with NPR1: TGA2 and TGA3 showed strong affinity for NPR1; TGA5 and TGA6 exhibited weaker affinity; and TGA1 and TGA4 displayed little or no detectable interaction with NPR1, respectively. Interestingly, the amino-termini of these factors were found to decrease their stability in yeast and differentially affect their apparent affinity toward NPR1. The interacting regions on NPR1 and the TGA factors were also defined. Each of four point mutations in NPR1 that disrupt SA signaling in Arabidopsis completely blocked interaction of NPR1 with TGA2 and TGA3. TGA2 and TGA3 were also found to bind the SA-responsive element of the Arabidopsis PR-1 promoter. These results directly link NPR1 to SA-induced PR-1 expression through members of the TGA family of transcription factors.

hsr203J is a tobacco gene whose activation is rapid, highly localized, and specific for incompatible interactions between tobacco and the bacterial pathogen Ralstonia solanacearum. The effect of other hypersensitive response (HR)-inducing pathogens and elicitors has been tested with transgenic plants containing the hsr203J promoter-GUS reporter gene fusion, and confirms the generality of the preferential inducibility of the hsr203J gene promoter during incompatible interactions: bacterial and viral pathogens inducing an HR in tobacco were able to induce the promoter fusion, as were inducers of HR-like responses such as harpin, elicitins, and PopA1 proteins. A tomato hsr203 homologous cDNA was isolated (Lehsr203) and used to examine the effect of avr gene products on the expression of such genes. Lehsr203 was shown to be rapidly and transiently induced in leaves of the tomato Cf-9 line, following Avr9 product infiltration, but not in those of the Cf-0 line. Among potential effectors of HR or resistance such as H2O2, salicylic acid, methyl jasmonate, and 2,6-dichloro-isonicotinic acid (INA), none is able to induce a significant increase in promoter activation. In contrast, heavy metals that cause leaf necrosis can trigger such an activation. In addition, hsr203-GUS fusion expression is detected in transgenic tobacco lines expressing the bO gene and exhibiting spontaneous HR-like lesions. Taken together, these results demonstrate a strong correlation between hsr203 and genetically controlled cell death in tobacco and tomato. The expression of this gene should be a useful marker for programmed cell death occurring in response not only to diverse pathogens, but also to diverse death-triggering extracellular agents.

Programmed cell death (PCD) is activated during the response of multicellular organisms to some invading pathogens. One of the key aspects of this process is the degradation of nuclear DNA which is thought to facilitate the recycling of DNA from dead cells. The PCD of tobacco plants (genotype NN) infected with tobacco mosaic virus (TMV) is accompanied by the induction of nuclease activities and the cleavage of nuclear DNA to fragments of about 50 kb. We examined the correlation between the increase in nuclease activities and the fragmentation of nuclear DNA during TMV- and bacteria-induced PCD in tobacco. We found that the increase in nuclease activities did not always correlate with fragmentation of nuclear DNA. Thus, in addition to pathogens that induce PCD, mechanical injury and infiltration of leaves with 1 M sucrose or bacteria that did not induce PCD also resulted in an increase in nuclease activities. Analysis of nuclease activities in total leaf extracts, nuclear extracts, and intercellular fluid (i.e., apoplast) revealed that at least four different nuclease activities are induced during PCD in tobacco; of these at least three appear to be secreted into the intercellular fluid. Although the latter were also induced in response to treatments that did not result in DNA fragmentation, they may function in the recycling of plant DNA during late stages of PCD when the integrity of the plasma membrane is compromised. This suggestion is supported by the finding that DNA degradation occurred late during TMV-induced PCD in tobacco. In addition, the finding of induced nuclease activities in the intercellular fluid raises the possibility that they may serve a protective function by degrading the DNA of invading pathogens.

We have isolated a cDNA clone from Glycine max, named SGA2, coding for a G alpha-subunit protein. The encoded polypeptide, SG alpha2, shows a molecular mass of 45 kDa and contains most of the conserved regions involved in guanine nucleotide binding and hydrolysis. Comparison at the nucleotide and amino acid sequence levels with the other plant G alpha's shows a high degree of conservation (>85% similarity). Phylogenetic analysis of these plant genes with the other G alpha's from different species clearly indicate that those proteins represent a new member of the heterotrimeric G-protein family, named Gp. Tissue localization of SGA2 transcripts in root, stem and leaf organs shows that this gene is widely expressed throughout the plant although it is most abundant in the vascular tissues of all these organs. Furthermore, the transcript is more abundant in young tissues and organ primordia than mature tissues. The high degree of sequence conservation among the plant G alpha's and the differences to other species of other kingdoms, suggest that plant G proteins may function in specialized signalling processes.

Nuclear localization serves as a regulatory mechanism in the activity of several transcription factors. KNOTTED-1 (Kn1) is a homeodomain protein likely to regulate vegetative development in maize. At least twelve genes related to Kn1 are known in maize and six in Arabidopsis. Ectopic expression of the maize, rice and Arabidopsis Kn1-related genes have been shown to alter cell fate determination. In this paper, we study the nuclear localization capabilities of the Kn1 homeodomain and the proximal amino acid residues (the ELK region) which is highly conserved among Kn1-related homeodomain proteins. The ELK homeodomain (ELK-HD) of Kn1 was fused to the reporter gene uidA encoding the bacterial enzyme beta-glucuronidase (GUS) and transformed into tobacco and onion cells. Quantitation of GUS activity in nuclear and total protein extracts from transgenic tobacco revealed a highly localized GUS activity in the nucleus for the ELK-HD/GUS fusion protein, as compared to the basal level of GUS activity in the nucleus for the GUS only protein. The ELK-HD/GUS transformants showed no unusual characteristics, thus indicating that expression of the putative Kn1 DNA-binding domain fused to GUS may be insufficient to create a dominant negative phenotype. Histochemical analysis of the onion epidermal cells transfected by particle bombardment demonstrated that greater than 50 % of the transformed onion epidermal cells showed higher levels of GUS staining in the nucleus relative to the cytoplasm. Deletion analysis of the ELK-HD revealed that the Kn1 homeodomain comprising the three predicted alpha-helices and the conserved ELK domain can each function independently as nuclear localization signals.

Activating sequence factor 1 (ASF-1) is a nuclear DNA-binding activity that is found in monocots and dicots. It interacts with several TGACG-containing elements that have been characterized from viral and T-DNA genes, the prototypes of which are the as-1 element of the CaMV 35S promoter and the ocs element from the octopine synthase promoter. This class of cis-acting elements can respond to auxin and salicylic acid treatments. Consistent with these observations, we have shown that ASF-1 can interact with promoter elements of an auxin-inducible tobacco gene GNT35, encoding a glutathione S-transferase. Characterization of the nuclear factors that make up ASF-1 activity in vivo will be an important step toward understanding this induction phenomenon. The TGA family of basic-leucine-zipper (bZIP) proteins are good candidates for the ASF-1 nuclear factor. However, there may be as many as seven distinct TGA genes in Arabidopsis, five of which have now been reported. In this study, we expressed the cDNAs that encode four of these five Arabidopsis TGA factors in vitro and compared their DNA-binding behavior using two types of TGACG-containing elements. With specific antisera prepared against three of the five known Arabidopsis TGA factors, we also investigated the relative abundance of these three proteins within the ASF-1 activities of root and leaf nuclear extracts. Our results indicate that these TGA factors bind to DNA with different degrees of cooperativity and their relative affinity toward as-1 also can differ significantly. The results of a supershift assay suggested that only one of the three TGA factors represented a significant component of nuclear ASF-1 activity. Arabidopsis TGA2 comprises approximately 33 and 50% of the ASF-1 activity detected in root and leaf nuclear extracts respectively. These results suggest that each member of the TGA factor family may be differentially regulated and that they may play different roles by virtue of their distinct DNA-binding characteristics. Furthermore, since transcripts for each of these factors can be detected in various plant tissues, post-transcriptional regulation may play an important part in determining their contribution to nuclear ASF-1 in a given cell type.

Light is an important environmental signal that can influence diverse developmental processes in plants. Many plant nuclear genes respond to light at the level of transcription initiation. GT-1 and GT2 are nuclear factors which interact with DNA sequences in many light-responsive gene promoters. cDNA clones which encode proteins with sequence binding specificities similar to those of these two factors have been isolated. They show significant amino acid sequence similarities within three closely spaced, putative alpha-helices that were predicted by secondary structure analysis but do not show significant homologies with any other reported DNA-binding protein. In this work, N- and C-terminal deletions of tobacco GT1a were generated by in vitro transcription and translation, and their DNA-binding activities and subunit structures were studied. The results suggest that the C-terminal domain of GT1a is critical for protein oligomerization, while a region predicted to contain four closely spaced alpha-helices is required for DNA binding. Direct chemical cross-linking and gel filtration analyses of full-length and truncated derivatives of GT1a suggest that this factor can exist in solution as a homotetramer and that oligomerization is independent of DNA binding. This study thus establishes two independent functional domains in this class of eukaryotic trans-acting factors. Possible implications of the multimeric nature of GT1a in relation to the known characteristics of light-responsive promoter architecture are discussed.

TGA1a is a well-characterized transcription factor that may mediate the root-specific and auxin-responsive expression of some plant genes. In tobacco, Southern blot and genomic cloning analyses have shown that TGA1a consists of at least four closely related genes. Since TGA1a belongs to the bZIP class of transcriptional factors, the protein products of the tobacco TGA1a family are likely to form hetero-dimers with each other in addition to the homo-dimers. In order to find a model plant system that may have less genomic complexity, we have now characterized a TGA1a-related gene (TGA3) from Arabidopsis thaliana. Southern blot analyses at high stringency suggest that Arabidopsis contains only one copy of TGA3 per haploid genome. However, low stringency Southern blot analyses with homologous and heterologous probes suggest that there is a multigene family of TGA1a-related genes present in Arabidopsis, of which TGA1, TGA2 and TGA3 are members. Although these gene members share a highly conserved bZIP region, they are not genes with high homologies at the nucleotide level. Similar to TGA1a of tobacco, TGA3 is most highly expressed in root tissues and recombinant TGA3 protein shows similar DNA-binding site specificity to that of TGA1a in vitro. Comparison of the genomic organization between TGA3 and the tobacco homologue PG13 reveals striking conservation in the sizes and positions of exons and introns in the region surrounding the bZIP domain.

The genes encoding the major light-harvesting antenna chlorophyll a/b-binding protein (Cab) of higher plants are regulated by light at the transcriptional level. In addition, their expression is largely restricted to photosynthetically competent organs such as leaves. A 268 bp fragment of the Cab-1 promoter from wheat functions as a light-responsive and organ-specific enhancer in transgenic tobacco. Using DNase I footprinting, four different regions have been mapped (Cab1-A, Cab1-B, Cab1-C and Cab1-D) in this fragment that bind to protein factors in tobacco nuclear extracts. Three of these regions (A, B and C) coincide with sequences that have been found to be functionally important from previous cis-element analyses. Synthetic tetramers of these three sites interact with different proteins in gel retardation assays. In addition, cross-competition analyses demonstrate that Cab1-C is likely to interact with ASF-2, a tobacco DNA-binding activity that binds to a conserved GATA element found in many dicot Cab promoters. In transgenic tobacco, a 95 bp fragment of the Cab-1 enhancer containing the A, B and C regions can confer leaf expression when fused upstream of a truncated derivative of the cauliflower mosaic virus (CaMV) 35S promoter. However, expression observed with this enhancer fragment in the promoter context of these studies does not appear to be significantly dependent on light. Similar results were obtained with synthetic tetramers of Cab1-A, -B or -C. These data thus suggest that the wheat Cab-1 enhancer contains at least three distinct elements that contribute to leaf-specific expression in transgenic tobacco. Interaction between factors binding to these positive elements and those that bind elsewhere in the Cab-1 enhancer may be necessary for light-responsive transcriptional activation.

Ribulose-1,5-bisphosphate carboxylase/oxygenase plays a key role in photosynthetic carbon fixation in higher plants. The small subunit of this chloroplast enzyme (rbcS), encoded by a family of nuclear genes, is regulated at the transcriptional level by light. Promoter analyses have previously identified the box II sequence as a cis element critical for the light-regulated expression of rbcS genes. Nuclear factor GT-1 binds specifically to this element and is one of the plant nuclear factors that has been detected and studied in great detail. Here we describe the cloning and characterization of a tobacco cDNA encoding a protein, designated B2F (Box II Factor), with similar binding specificity and mobility in gel retardation assays as nuclear GT-1. Steady state levels of mRNA encoding B2F do not appear to be regulated by light; this is consistent with the previous observation that nuclear GT-1 activity is present in extracts from both light-grown and dark-adapted plants. Sequence comparison with another plant trans-acting factor, GT-2, which binds to a GT-like element in the rice phytochrome promoter, shows striking homology in three putative alpha-helices that may be involved in DNA binding.

A conserved 21-base pair element, designated as hex-1, located between -180 and -160 of the wheat histone H3 promoter, is known to interact with two tobacco nuclear factors, activating sequence factor 1 and hex-1-specific binding factor. We have shown previously that a mutant sequence (hex-3), which differs from hex-1 by three base pairs, can no longer bind these two factors significantly. In the present work, we examined the functional characteristics of these two sequences in transgenic tobacco. Surprisingly, we found that a tetramer of hex-3, but not of hex-1, confers high level expression in mature seeds. Expression of this synthetic promoter rapidly diminishes upon germination but can be reactivated in young seedlings and mature leaves by desiccation, NaCl, or the phytohormone abscisic acid (ABA). Treatment with auxin or cytokinin has no apparent effect on the expression. Since the endogenous ABA level of plant cells is known to increase upon water stress, our data suggest that hex-3, the mutated hex-1 sequence, is an abscisic acid-responsive element (abre). We propose that a tobacco nuclear factor, distinct from activating sequence factor 1 and hex-1-specific binding factor, interacts with this sequence and is involved in mediating the effects of ABA and water stress on gene expression.

We have used DNase I footprinting to characterize nuclear factors that bind to the light-responsive promoter of pea rbcS-3A, one member of the gene family encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. A sequence-specific binding activity, designated 3AF1, binds to an AT-rich sequence present at the -45 region of the rbcS-3A promoter. A tetramer of the 3AF1 binding site, designated as Box VI, can form multiple complexes with tobacco leaf and root nuclear extracts. Mutations of 3 base pairs in Box VI severely reduce DNA-protein complex formation in vitro. The wild-type Box VI tetramer, but not the mutant tetramer, is active in transgenic tobacco plants when placed upstream of the cauliflower mosaic virus 35S promoter truncated at -90. These results correlate binding of 3AF1 to the in vivo function of Box VI. The Box VI tetramer/35S chimeric construct confers expression in diverse cell types and organs and its activity is not dependent on light. By using the Box VI tetramer as a probe to screen a cDNA expression library, we have obtained a putative cDNA clone for the 3AF1 DNA-binding activity. Lysogen extracts of Escherichia coli expressing the cDNA clone give sequence-specific complexes with Box VI. The deduced amino acid sequence of the protein encoded by the cDNA contains two stretches of about 100 residues that are 80% homologous. Moreover, in each of the two repeats, there is an arrangement of histidines and cysteines, which may be related to the two known types of zinc-finger motifs found in many DNA-binding proteins. Consistent with the expectation that metal coordination plays an important role in DNA binding by this protein, we found that 1,10-phenanthroline can abolish the formation of DNA-protein complexes. Interestingly, we found that the same treatment did not abolish the DNA binding activity of 3AF1 in crude nuclear extracts of tobacco. These data indicate that the nuclear 3AF1 activity is likely due to multiple DNA-binding proteins all interacting with Box VI in vitro. RNA gel blot analysis shows that multiple transcripts homologous to this cDNA clone are expressed in different tobacco organs.

Light-dependent expression of rbcS, the gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase, which is the key enzyme involved in carbon fixation in higher plants, is regulated at the transcriptional level. Sequence analysis of the gene has uncovered a conserved GT motif in the -150 to -100 region of many rbcS promoters. This motif serves as the binding site of a nuclear factor, designated GT-1. Analysis of site-specific mutants of pea rbcS-3A promoter demonstrated that GT-1 binding in vitro is correlated with light-responsive expression of the rbcS promoter in transgenic plants. However, it is not known whether factors other than GT-1 might also be required for activation of transcription by light. A synthetic tetramer of box II (TGTGTGGTTAATATG), the GT-1 binding site located between -152 to -138 of the rbcS-3A promoter, inserted upstream of a truncated cauliflower mosaic virus 35S promoter is sufficient to confer expression in leaves of transgenic tobacco. This expression occurs principally in chloroplast-containing cells, is induced by light, and is correlated with the ability of box II to bind GT-1 in vitro. The data show that the binding site for GT-1 is likely to be a part of the molecular light switch for rbcS activation.

The 35S promoter of the cauliflower mosaic virus (CaMV) contains a tandem repeat of the sequence TGACG in the region -83 to -63. This 21-base pair (bp) sequence, called as-1, is involved in root expression of the 35S promoter. When inserted in a promoter of a gene expressed specifically in photosynthetic tissues, as-1 confers high level expression in roots. We have described a factor, ASF-1, that binds specifically to as-1 in vitro. There is a good correlation between ASF-1 binding affinity to as-1 related sequences in vitro and the function of these sequences in vivo. These results strongly suggest that ASF-1 is responsible for the function of as-1. Here we report the isolation of tobacco complementary DNA clones encoding two TGACG-sequence-specific binding-proteins (TGA1a and TGA1b). Sequence analysis of the cDNA clones shows that both proteins contain a basic region that shows high homology to a stretch of basic amino acids in the nuclear factors CREB, GCN4, and c-Jun to a 'leucine-zipper' region. On the basis of binding specificity we propose TGA1a to be a good candidate for ASF-1.

A photoautotrophic suspension culture of soybeans was found to exhibit light-dependent expression of the genes encoding the major chlorophyll a- and b-binding protein (CAB). The expression was mediated by phytochrome, since it was induced by red light and reversed by far-red light. The maximal level as well as the kinetics of the induction were comparable between the suspension culture and soybean seedlings. Using this cell culture, we addressed the question of whether a calcium- and/or calmodulin-mediated step is involved in the signal transduction process between phytochrome and CAB expression. We found that W-7, a potent calmodulin antagonist, severely attenuated the induction of CAB mRNA by light, whereas W-5, a weak calmodulin antagonist, had little effect. Control experiments demonstrated that W-7 treatment did not block the induction of hsp-75 by heat shock. The addition of ionomycin, a calcium ionophore, induced a low level of CAB mRNA accumulation in the dark which could be further enhanced by light treatment. We propose that calmodulin activation by light is necessary but not sufficient to induce maximal CAB expression.

The genes coding for the light-harvesting chlorophyll a/b-binding protein of photosystem II (cab) in wheat are regulated by phytochrome and an endogenous circadian clock. The regulation by these factors has been shown to be at the transcriptional level. Here we show that the phase of cab gene expression is reset by light to dark transition of the previous day. The time of light to dark transition is linearly related to the time of maximal cab gene expression under free running conditions in darkness. We propose that in nature the onset of darkness resets the phase of the circadian clock, which regulates the timing of cab gene expression, so as to anticipate the time of sunrise in the following day.

The photoreduction of soluble plastocyanin in a homologous reconstituted system requires a resolved Photosystem II preparation and the cytochrome b6-f complex from spinach chloroplasts. Oxygen evolution is linked quantitatively to plastocyanin reduction in the reconstituted system, indicating that water is the electron donor for the photoreduction. The photoreduction is sensitive to inhibitors which specifically interact with the Photosystem II preparation [DCMU: 3-(3,4-dichlorophenyl)-1,1-dimethylurea] as well as those which interact with the cytochrome complex (DNP-INT: 2-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether; and DBMIB: 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone). The plastocyanin requirement in the reconstituted system indicated a Km of 4.6 microM and a Vmax of 4.2 mumols plastocyanin reduced per nmol cytochrome f per h. Inhibition curves for DNP-INT and DBMIB show that both are effective inhibitors (50% inhibition at a ratio of inhibitor to cytochrome f of 1.0). The rate of reduction of plastocyanin was found to be stimulated by cations. This dependency was localized in the transfer of electrons from the Photosystem II preparation to the cytochrome complex. These results are considered in terms of organizational aspects of integral protein complexes in thylakoid membranes.

The cytochrome b6-f complex from spinach thylakoids has been reconstituted with an oxygen-evolving Photosystem II (PSII) preparation isolated from the same source to give oxygenic plastocyanin reductase activity. We observe that (i) mixing of the two complexes in concentrated form prior to dilution with the assay medium is necessary for optimal reconstitution of activity; (ii) incubation for longer times after dilution can also give substantial reconstitution if the two complexes are added separately to the assay mixture; (iii) either monovalent or divalent cations are required for optimum activity in the reconstituted system; (iv) titration of the cytochrome complex with varying amounts of the PSII complex gave a saturation of the plastocyanin reduction activity at a cytochrome complex/PSII ratio of 3-4; (v) kinetic analysis of plastocyanin photoreduction by Photosystem II shows nonlinearity, while first-order reduction kinetics are observed with duroquinol as electron donor; and (vi) as the concentration of plastocyanin is increased, the half-time of the reduction increases. These observations are considered in terms of a functional association between PSII and the cytochrome b6-f complex in this reconstituted system, and the relevance of these observations to the situation in vivo is discussed.

Photosystem I (PSI) and photosystem II (PSII) complexes have been isolated from stacked spinach thylakoid membranes that had been treated with varying amounts of glutaraldehyde. The concentrations of cytochrome f, Q, and P700 have been determined by spectrophotometric methods. It was found that at low concentrations of glutaraldehyde, the amount of cytochrome f associated with either PSII or PSI increased significantly while the amounts of Q and P700 stayed relatively constant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analyses indicated the presence of cytochrome f and other components of the cytochrome b6-f complex in the PSII and PSI preparations after glutaraldehyde treatment, but no intermolecular cross-linked polypeptides could be detected. Solubilization of the cytochrome b6-f complex was also inhibited after thylakoid membranes were treated with low concentrations of glutaraldehyde. These results are discussed in relation to current models for the organization of the membrane complexes, and relate to the location of the cytochrome b6-f complex in appressed and nonappressed membrane regions of thylakoids.

We have investigated the effects of protein synthesis inhibitors on light-induced expression of two plant nuclear genes, Cab and rbcS, in wheat, pea and transgenic tobacco. Light activation of these two genes is very sensitive to cycloheximide, an inhibitor of cytoplasmic protein synthesis but not to chloramphenicol, an inhibitor of organellar protein synthesis. Studies with chimeric gene constructs in transgenic tobacco seedlings show that cycloheximide exerts its effect at the transcriptional level. As a control, we show that the expression of the cauliflower mosaic virus (CaMV) 35S promoter is enhanced by cycloheximide treatment, irrespective of the coding sequence used. Escape-time analyses with green wheat seedlings show that the cycloheximide block for Cab gene expression is after the primary signal transduction step linked to phytochrome photoconversion. Our results suggest that phytochrome activation of Cab and rbcS is mediated by a labile protein factor(s) synthesized on cytoplasmic ribosomes.