Phosphorylation of tobacco eukaryotic translation initiation factor 4A upon pollen tube germination.

Eukaryotic translation initiation factor eIF-4A is a member of the DEAD box family of RNA helicases and RNA-dependent ATPases. In tobacco, eIF-4A is encoded by a gene family with one isoform, eIF-4A8, being exclusively expressed in pollen. This pollen-specific isoform is a candidate for mediating translational control in the developing gametophyte. Here we show that eIF-4A is barely phosphorylated in mature pollen, but during pollen tube germination, two isoforms of eIF-4A become phosphorylated. Phosphoamino acid analysis indicated phosphorylation of threonine. In order to determine whether pollen-specific eIF-4A8 is among the phosphorylated isoforms, we raised transgenic tobacco plants overexpressing eIF-4A8 containing a histidine tag. Hereby, we could show that indeed eIF-4A8 is modified through phosphorylation. The biological relevance of the phosphorylation of eIF-4A is discussed.

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.

Aldehyde dehydrogenase in tobacco pollen.

Acetaldehyde is one of the intermediate products of ethanolic fermentation, which can be reduced to ethanol by alcohol dehydrogenase (ADH). Alternatively, acetaldehyde can be oxidized to acetate by aldehyde dehydrogenase (ALDH) and subsequently converted to acetyl-CoA by acetyl-CoA synthetase (ACS). To study the expression of ALDHs in plants we isolated and characterized a cDNA coding for a putative mitochondrial ALDH (TobAldh2A) in Nicotiana tabacum. TobALDH2A shows 54-60% identity at the amino acid level with other ALDHs and shows 76% identity with maize Rf2, a gene involved in restoration of male fertility in cms-T maize. TobAldh2A transcripts and protein were present at high levels in the male and female reproductive tissues. Expression in vegetative tissues was much lower and no induction by anaerobic incubation was observed. This suggests that TobALDH expression is not part of the anaerobic response, but may have another function. The use of specific inhibitors of ALDH and the pyruvate dehydrogenase (PDH) complex indicates that ALDH activity is important for pollen tube growth, and thus may have a function in biosynthesis or energy production.

Aerobic fermentation in tobacco pollen.

We characterized the genes coding for the two dedicated enzymes of ethanolic fermentation, alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC), and show that they are functional in pollen. Two PDC-encoding genes were isolated, which displayed reciprocal regulation: PDC1 was anaerobically induced in leaves, whereas PDC2 mRNA was absent in leaves, but constitutively present in pollen. A flux through the ethanolic fermentation pathway could be measured in pollen under all tested environmental and developmental conditions. Surprisingly, the major factor influencing the rate of ethanol production was not oxygen availability, but the composition of the incubation medium. Under optimal conditions for pollen tube growth, approximately two-thirds of the carbon consumed was fermented, and ethanol accumulated into the surrounding medium to a concentration exceeding 100 mM.

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.

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.

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.

Localization and conditional redundancy of regulatory elements in rbcS-3A, a pea gene encoding the small subunit of ribulose-bisphosphate carboxylase.

Expression of the pea rbcS-3A gene, one of a family of genes encoding the small subunit of ribulose-bisphosphate carboxylase [EC 4.1.1.39], is regulated by light and is restricted to chloroplast-containing cells. We analyzed the effects of light and development on rbcS-3A expression in transgenic plants. Two highly conserved sequences ("boxes" II and III) around nucleotide position -150 (relative to the transcription initiation site, +1) are required for rbcS-3A expression. The so-defined positive elements overlap with previously identified negative light-regulatory elements. In the case of box II, which has sequence similarity to the core enhancer motif of simian virus 40, a GG----CC transversion is sufficient to abolish expression. The effect of mutations in boxes II and III can only be measured when sequences upstream of -170 are removed, and because sequences both 5' and 3' of -170 can direct light-regulated and organ-specific expression. This implies that there is a redundancy of cis-acting elements in the 5' noncoding region of rbcS-3A. However, we show that the sequences upstream of -170 are dispensable only in the mature leaves of a green plant. In contrast, in the young, expanding leaves at the top of a green plant, as well as in seedlings, the distal elements are required for high-level expression. Therefore, redundancy is not absolute, and the requirements for rbcS-3A expression change during plant development.

Sequences in the pea rbcS-3A gene have homology to constitutive mammalian enhancers but function as negative regulatory elements.

The pea rbcS-3A gene, which codes for a key component of the photosynthetic machinery, requires light for its expression. Analysis of chimeric constructs in transgenic tobacco plants has shown that a 280-bp fragment from the 5' noncoding region can act as a light-inducible transcriptional enhancer. Further characterization of this enhancer identifies a 58-bp sequence containing two regulatory elements that can decrease transcription in the dark. One has a high degree of homology to the SV40 core enhancer, the other to the adenovirus 5 E1A enhancer and the constitutive part of the human interferon-beta gene enhancer. Deletion of the 58-bp sequence uncovers additional light-responsive elements (LREs) located further upstream and downstream.