Organ-specific and light-induced expression of plant genes.

Light plays a pivotal role in the development of plants. The photoregulation of plant genes involves recognition of light quality and quantity by phytochrome and other light receptors. Two gene families, rbcS and Cab, which code for abundant proteins active in photosynthesis, the small subunit of ribulose bisphosphate carboxylase and the chlorophyll a/b binding protein, show a 20-to 50-fold increase in transcript abundance in the light. Analyses in calli and transgenic plants of deletions of the rbcS gene and of chimeric constructions has allowed localization of two regions involved in light-induced transcription. One element is confined to a 33-base pair region surrounding the TATA box. In addition, an enhancer-like element contained within a 240-base pair fragment can confer phytochrome-induced transcription and organ specificity on nonregulated promoters.

The Pea rbcS-3A Promoter Mediates Light Responsiveness but not Organ Specificity.

The expression of the pea rbcS-3A gene is responsive to light and restricted to chloroplast-containing cells. Previously, we identified transcriptional elements between -410 and -50 relative to the transcription start site as being sufficient for this regulated expression. Here we investigate the role of the promoter region (-50 to +15) in rbcS-3A expression. We demonstrate an interaction between the promoter and upstream sequences located between -189 and -166, which is important for high level expression. In addition we show that, when the rbcS promoter is driven by a weak test enhancer, it can confer light-responsive but not organ-specific expression upon a bacterial reporter gene.

Auxin and phyllotaxis.

Our understanding of phyllotaxis is still largely based on surgical and pharmacological experiments carried out before 1970. Recent experiments implicate the plant hormone auxin in the regulation of phyllotaxis. A recent paper shows how the polar auxin transport mutant, pin1-1, which fails to make flowers, affects the expression of well known meristem genes. This work opens the door for the genetic analysis of phyllotaxis.

Highly conserved genes coding for eukaryotic translation initiation factor eIF-4A of tobacco have specific alterations in functional motifs.

Eukaryotic translation initiation factor eIF-4A is an ATP-dependent RNA helicase that is required for the binding of mRNA to ribosomes. Plant eIF-4A-like proteins are highly homologous to eIF-4As from yeast, mouse and Drosophila melanogaster. The pattern of intron-exon boundaries in eIF-4A-like genes are conserved within tobacco, but are not conserved with other organisms. Fixed spacings between the functionally important sequence motifs, GKT-PTRELA (72 bp), DEAD-SAT (81 bp) and SAT-HRIGR (426 bp), are conserved between plants, mouse, Drosophila and yeast.

Characterization of nodule-specific cDNA clones from Sesbania rostrata and expression of the corresponding genes during the initial stages of stem nodules and root nodules formation.

We have constructed a Sesbania rostrata stem nodule-specific cDNA library. By screening with heterologous probes from pea and soybean, we have isolated several nodulin cDNA clones. On the basis of nucleotide and amino acid sequence homology, two nearly full-length cDNA clones coding for two different leghemoglobin-like proteins have been identified. The inserts of two other clones reveal a high degree of amino acid sequence homology (81% and 72%) to the early nodulin Enod2 from soybean; the characteristic heptapeptide repeat units PPHEKPP and PPYEKPP of the soybean Enod2 are conserved in the proteins encoded by these Sesbania cDNA clones. The time course of Enod2 and leghemoglobin mRNA appearance during the formation of stem nodules and root nodules on S. rostrata was analyzed by northern blot hybridization. Significant differences were found for the initiation of mRNA accumulation of these nodulins between S. rostrata and soybean.

Cloning of a third nitrate reductase gene from the cyanobacterium Anacystis nidulans R2 using a shuttle cosmid library.

A strategy for gene cloning in the cyanobacterium Anacystis nidulans R2 was developed which made use of a gene library constructed in a shuttle cosmid vector. The method involved phenotypic complementation of mutants with pooled cosmid DNA. The development of the procedure and its application to the cloning of a third gene involved in nitrate reduction are described.

A new approach for molecular cloning in cyanobacteria: cloning of an Anacystis nidulans met gene using a Tn901-induced mutant.

A new strategy for molecular cloning in the cyanobacterium Anacystis nidulans R-2 is described. This strategy involved the use of a transposon and was developed for the cloning of a gene encoding methionine biosynthesis. A met::Tn901 mutant was isolated. Chromosomal DNA fragments were cloned in the Escherichia coli plasmid vector pACYC184. A recombinant plasmid carrying the inactivated met::Tn901 gene was selected after transformation to E. coli. The cloned met::Tn901 DNA fragment was used as a probe to select the corresponding A. nidulans R-2 wild-type met gene from a gene library prepared in E. coli, using the newly constructed shuttle cosmid vector pPUC29. When transformed into A. nidulans Met- mutants, this cloned gene allowed the mutants to grow prototrophically.

Minimal sequence requirements for the regulated expression of rbcS-3A from Pisum sativum in transgenic tobacco plants.

RbcS-3A, the most highly expressed member of the pea multigene family encoding the small subunit of ribulose 1,5-bisphosphate carboxylase, is expressed in a light-dependent and organ-specific manner. In order to further delineate the sequences which mediate this complex pattern of regulation, putative regulatory sequences were assayed for function in transgenic tobacco plants in the context of an inactive 5' deleted rbcS-3A test gene. We have identified a minimal functional unit of 58 bp which is able to confer organ-specific transcriptional activity. It contains two sequences conserved among the pea rbcS family members, namely box II (-151 to -138; GTGTGGTTAATATG) and box III (-125 to -114; ATCATTTTCACT). These sequences bind the nuclear factor termed GT-1 in vitro. Substitution mutations within this 58 bp element have demonstrated that sequences upstream of, or located between, boxes II and III are not required for the transcriptional activity conferred by this element. Distance and orientation of these sequences from the gene are not critical for activity within the limits tested. DNA fragments upstream of nucleotide -170 of rbcS-3A that contain other GT-1 binding sites can also confer regulated expression upon the rbcS-3A promoter deleted to -50. Multimers of individual motifs, namely four tandem copies of boxes II and III, are unable to drive expression of the deleted promoter. These observations suggest that while GT-1 binding is necessary for promoter activity it is by itself not sufficient.

Color and scent: how single genes influence pollinator attraction.

A major function of angiosperm flowers is the recruitment of animal pollinators that serve to transfer pollen among conspecific plants. Distinct sets of floral characteristics, called pollination syndromes, are correlated with visitation by specific groups of pollinators. Switches among pollination syndromes have occurred in many plant families. Such switches must have involved coordinated changes in multiple traits and multiple genes. Two well-studied floral traits affecting pollinator attraction are petal color and scent production. We review current knowledge about the biosynthetic pathways for floral color and scent production and their interaction at the genetic and biochemical levels. A key question in the field concerns the genes that underlie natural variation in color and scent and how such genes affect pollinator preference, reproductive isolation, and ultimately speciation.

Differential expression of genes encoding the hypusine-containing translation initiation factor, eIF-5A, in tobacco.

Two Nicotiana plumbaginifolia cDNA clones, NeIF-5A1 and NeIF-5A2, encoding eukaryotic translation initiation factor eIF-5A (formerly called eIF-4D) were cloned by heterologous screening with Dictyostelium and human eIF-5A probes. eIF-5A is the only protein known to contain a unique amino acid modification, hypusine. Comparison of the Nicotiana deduced amino acid sequences with those of other eIF-5A polypeptides reveals conservation throughout the coding sequence, especially in the region of the hypusine residue. Transcript analysis reveals that NeIF-5A1 is preferentially expressed in photosynthetic tissues, while NeIF-5A2 is constitutively expressed in all plant tissues examined. A polyclonal antibody was raised against NeIF-5A1 overexpressed in E. coli. NeIF-5A1 antiserum crossreacts with an 18 kDa polypeptide doublet in all tobacco tissues examined. At least one polypeptide of ca. 18 kDa from a diversity of higher and lower plants crossreacts with NeIF-5A1 antiserum.

Long-term anoxia tolerance. Multi-level regulation of gene expression in the amphibious plant Acorus calamus L.

Acorus calamus is a monocotyledonous wetland plant that can withstand extremely long periods of anoxia. We have investigated the expression of genes coding for pyruvate decarboxylase (Pdc), alcohol dehydrogenase (Adh), and fructose-1,6-bisphosphate aldolase (Ald) during periods of anoxia ranging from 2 h to 2 months. Upon anoxic incubation, Pdc mRNA levels peak at 6 h, followed by Adh and Ald, which peak at 12 and 72 h, respectively. Subsequently, the mRNA levels of all three genes decline within days to low levels. In contrast, alcohol dehydrogenase (ADH) protein levels increase steadily for at least a week and then remain constant. Native gel electrophoresis demonstrates the presence of two sets of ADH isozymes, one present constitutively, the other enhanced during anoxia. Translation initiation factor 4A protein levels, used as a control, remain constant during 2 months of anoxia. The results suggest that A. calamus has developed a complex anaerobic response consisting of differential regulation of transcription, translation, and posttranslational processes.

Aerobic fermentation during tobacco pollen development.

In vegetative organs of plants, the metabolic switch from respiration to fermentation is dictated by oxygen availability. The two genes dedicated to ethanolic fermentation, pyruvate decarboxylase and alcohol dehydrogenase, are induced by oxygen deprivation and the gene products are active under oxygen stress. In pollen, these two genes are expressed in a stage-specific manner and transcripts accumulate to high levels, irrespective of oxygen availability. We have examined the expression pattern of pyruvate decarboxylase and alcohol dehydrogenase at the protein level in developing pollen and show that the active proteins are localized to the gametophytic tissue and begin to accumulate at microspore mitosis. A flux through the ethanolic fermentation pathway could already be detected very early in pollen development, occurring in all stages from premeiotic buds to mature pollen. This flux was primarily controlled not by oxygen availability, but rather by sugar supply. At a high rate of sugar metabolism, respiration and fermentation took place concurrently in developing and germinating pollen. We propose that aerobic fermentation provides a shunt from pyruvate to acetyl-CoA to accommodate the increased demand for energy and biosynthetic intermediates during pollen development and germination. A possible undesirable side-effect is the potential accumulation of toxic acetaldehyde. Our results support a model for cms-T-type male sterility in maize, in which degeneration of the tapetum is caused by the toxic effects of acetaldehyde on mitochondria weakened by the presence of the URF13 protein.

A ubiquitously expressed MADS-box gene from Nicotiana tabacum.

Experiments in Antirrinum majus, Arabidopsis thaliana and Petunia hybrida have demonstrated that putative transcription factors of the so-called MADS-box family play an important role in determining floral organ identity. Such regulatory genes are transiently expressed in small numbers of cells in the floral apex. Here we describe the isolation of a cDNA from Nicotiana tabacum coding for a MADS-box protein which is expressed in both the floral and vegetative organs of the plant.

Ethanolic fermentation in transgenic tobacco expressing Zymomonas mobilis pyruvate decarboxylase.

During oxygen limitation in higher plants, energy metabolism switches from respiration to fermentation. As part of this anaerobic response the expression of genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) is strongly induced. In addition there is ample evidence for post-translational regulation. In order to understand this multi-level regulation of the anaerobic response, we provided tobacco with the constitutive capacity of ethanolic fermentation by expressing a PDC gene derived from the obligate anaerobe Zymomonas mobilis. The protein accumulated to high levels and was active in an in vitro assay. During the first 2-4 h of anoxia, acetaldehyde accumulated to 10- to 35-fold and ethanol to 8- to 20-fold higher levels than in wild-type. Under normoxic conditions no accumulation of acetaldehyde and ethanol could be measured. Instead, the two products may be immediately re-metabolized in tobacco leaf tissue. We show that aerobic fermentation takes place when the respiratory system is inhibited. Although these conditions enhance ethanolic fermentation under normoxia, they fail to increase ADH transcript levels. These results indicate that anaerobic transcription is triggered not by the metabolic consequences of oxygen limitation, but directly through an oxygen-sensing system.

A pollen-specific DEAD-box protein related to translation initiation factor eIF-4A from tobacco.

A pollen-specific sequence, NeIF-4A8, has been isolated from a cDNA library from mature pollen of Nicotiana tabacum cv. Samsun. NeIF-4A8 is a full-length cDNA whose deduced amino acid sequence exhibits high homology to the eucaryotic translation initiation factor eIF-4A from mouse, Drosophila and tobacco. eIF-4A is an RNA helicase which belongs to the supergene family of DEAD-box proteins. Northern blot analysis with a gene-specific probe showed strict anther-specific expression of NeIF-4A8 starting at microspore mitosis. With antibodies raised against tobacco eIF-4A the presence of abundant eIF-4A-related proteins in developing anthers and pollen grains was demonstrated. The genomic analysis shows that the coding region is split by three introns whereas a large, fourth intron is situated in the 5'-untranslated region. A promoter construct with 2137 bp of upstream sequence fused to the GUS reporter gene was used to confirm that the expression is confined to the haploid cells within the anther. NeIF-4A8 is a prime candidate formediating translational control in the developing male gametophyte.

Auxin regulates the initiation and radial position of plant lateral organs.

Leaves originate from the shoot apical meristem, a small mound of undifferentiated tissue at the tip of the stem. Leaf formation begins with the selection of a group of founder cells in the so-called peripheral zone at the flank of the meristem, followed by the initiation of local growth and finally morphogenesis of the resulting bulge into a differentiated leaf. Whereas the mechanisms controlling the switch between meristem propagation and leaf initiation are being identified by genetic and molecular analyses, the radial positioning of leaves, known as phyllotaxis, remains poorly understood. Hormones, especially auxin and gibberellin, are known to influence phyllotaxis, but their specific role in the determination of organ position is not clear. We show that inhibition of polar auxin transport blocks leaf formation at the vegetative tomato meristem, resulting in pinlike naked stems with an intact meristem at the tip. Microapplication of the natural auxin indole-3-acetic acid (IAA) to the apex of such pins restores leaf formation. Similarly, exogenous IAA induces flower formation on Arabidopsis pin-formed1-1 inflorescence apices, which are blocked in flower formation because of a mutation in a putative auxin transport protein. Our results show that auxin is required for and sufficient to induce organogenesis both in the vegetative tomato meristem and in the Arabidopsis inflorescence meristem. In this study, organogenesis always strictly coincided with the site of IAA application in the radial dimension, whereas in the apical-basal dimension, organ formation always occurred at a fixed distance from the summit of the meristem. We propose that auxin determines the radial position and the size of lateral organs but not the apical-basal position or the identity of the induced structures.

Definition of constitutive gene expression in plants: the translation initiation factor 4A gene as a model.

The NeIF-4A10 gene belongs to a family of at least ten genes, all of which encode closely related isoforms of translation initiation factor 4A. The promoter region of NeIF-4A10 was sequenced, and four mRNA 5' ends were determined. Deletions containing 2750, 689 and 188 bp of untranscribed upstream DNA were fused to the GUS reporter gene and introduced into transgenic tobacco. The three constructs mediated GUS expression in all cells of the leaf, stem and shoot apical meristem. Control experiments using in situ hybridization and tissue printing indicated that the observed GUS expression matches the expression patterns of NeIF-4A mRNA and protein. This detailed analysis at the level of mRNA, protein and reporter gene expression shows that NeIF-4A10 is an ideal constitutively expressed control gene. We argue that inclusion of such a control gene in experiments dealing with specifically expressed genes is in many cases essential for the correct interpretation of observed expression patterns.

Divergent genes for translation initiation factor eIF-4A are coordinately expressed in tobacco.

Three cDNA clones coding for eukaryotic translation initiation factor 4A, eIF-4A, were isolated from a Nicotiana plumbaginifolia root cDNA library by heterologous screening. The clones comprise two distinct gene classes as two clones are highly similar while the third is divergent. The genes belong to a highly conserved gene family, the DEAD box supergene family, although the divergent clone contains a DESD box rather than the characteristic DEAD box. The two clones are representatives of separate small multigene families in both N. plumbaginifolia and N. tabacum. Representatives of each family are coordinately expressed in all plant organs examined. The 47 kD polypeptide product of one clone, overexpressed in E. coli, crossreacts immunologically with a rabbit reticulocyte eIF-4A polyclonal antibody. Taken together the data suggest that the two Nicotiana eIF-4A genes encode translation initiation factors. The sequence divergence and the coordinate expression of the two Nicotiana eIF-4A families provide an excellent system to determine if functionally distinct eIF-4A polypeptides are required for translation initiation in plants.

The patterns of gene expression in the tomato shoot apical meristem.

In this paper, we describe the synthesis of a cDNA library from the vegetative shoot apical meristem and the analysis of clones selected from it. Using in situ hybridization, we characterized the patterns of expression of these genes in the tomato shoot apical meristem, as well as the patterns obtained from other sources. The results from the analysis of 15 cDNAs indicated the following six main patterns of gene expression in the shoot apical meristem: overall expression, zero expression, expression limited to the epidermis, expression excluded from the epidermis, punctate expression, and expression elevated in the flanks of the meristem. The patterns observed and the nature and number of the genes showing these patterns necessitate a reinterpretation of the models of meristem structure and function. In particular, the data suggest a compartmentation within the shoot apical meristem based on the spatial expression of particular subsets of genes. This paper also reports on the specific and precise criteria essential for the correct identification of meristem-specific gene expression. The data give new insight into the molecular organization of the shoot apical meristem and provide the framework for a detailed dissection of the factors controlling this organization.