A pollen-specific RALF from tomato that regulates pollen tube elongation.

Rapid Alkalinization Factors (RALFs) are plant peptides that rapidly increase the pH of plant suspension cell culture medium and inhibit root growth. A pollen-specific tomato (Solanum lycopersicum) RALF (SlPRALF) has been identified. The SlPRALF gene encodes a preproprotein that appears to be processed and released from the pollen tube as an active peptide. A synthetic SlPRALF peptide based on the putative active peptide did not affect pollen hydration or viability but inhibited the elongation of normal pollen tubes in an in vitro growth system. Inhibitory effects of SlPRALF were detectable at concentrations as low as 10 nm, and complete inhibition was observed at 1 mum peptide. At least 10-fold higher levels of alkSlPRALF, which lacks disulfide bonds, were required to see similar effects. A greater effect of peptide was observed in low-pH-buffered medium. Inhibition of pollen tube elongation was reversible if peptide was removed within 15 min of exposure. Addition of 100 nm SlPRALF to actively growing pollen tubes inhibited further elongation until tubes were 40 to 60 mum in length, after which pollen tubes became resistant to the peptide. The onset of resistance correlated with the timing of the exit of the male germ unit from the pollen grain into the tube. Thus, exogenous SlPRALF acts as a negative regulator of pollen tube elongation within a specific developmental window.

Mitochondrial retrograde regulation in plants.

Plant cells must react to a variety of adverse environmental conditions that they may experience on a regular basis. Part of this response centers around (1) ROS as damaging molecules and signaling molecules; (2) redox status, which can be influenced by ROS production; and (3) availability of metabolites. All of these are also likely to interface with changes in hormone levels [Desikan, R., Hancock, J., Neill, S., 2005. Reactive oxygen species as signalling molecules. In: Smirnoff, N. (ed.), Antioxidants and reactive oxygen species in plants. Blackwell Pub. Ltd., Oxford, pp. 169-196; Kwak, J.M., Nguyen, V., Schroeder, J.I., 2006. The role of reactive oxygen species in hormonal responses. Plant Physiol. 141, 323-329]. Each of these areas can be strongly influenced by changes in mitochondrial function. Such changes trigger altered nuclear gene expression by a poorly understood process of mitochondrial retrograde regulation (MRR), which is likely composed of several distinct signaling pathways. Much of what is known about plant MRR centers around the response to a dysfunctional mtETC and subsequent induction of genes encoding proteins involved in recovery of mitochondrial functions, such as AOX and alternative NAD(P)H dehydrogenases, and genes encoding enzymes aimed at regaining ROS level/redox homeostasis, such as glutathione transferases, catalases, ascorbate peroxidases and superoxide dismutases. However, as evidence of new and interesting targets of MRR emerge, this picture is likely to change and the complexity and importance of MRR in plant responses to stresses and the decision for cells to either recover or switch into programmed cell death mode is likely to become more apparent.

Comparison of intact Arabidopsis thaliana leaf transcript profiles during treatment with inhibitors of mitochondrial electron transport and TCA cycle.

Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Mitochondrial reactive oxygen species (mtROS) are a MRR signaling component, but whether all MRR requires ROS is not established. Inhibition of the cytochrome respiratory pathway by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA), each of which initiates MRR, can increase ROS production in some plant cells. We found that for AA and MFA applied to leaves of soil-grown Arabidopsis thaliana plants, ROS production increased with AA, but not with MFA, allowing comparison of transcript profiles under different ROS conditions during MRR. Variation in transcript accumulation over time for eight nuclear encoded mitochondrial protein genes suggested operation of both common and distinct signaling pathways between the two treatments. Consequences of mitochondrial perturbations for the whole transcriptome were examined by microarray analyses. Expression of 1316 and 606 genes was altered by AA and MFA, respectively. A subset of genes was similarly affected by both treatments, including genes encoding photosynthesis-related proteins. MFA treatment resulted in more down-regulation. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from AA or MFA inhibition were most similarly affected by biotic stresses such as pathogens. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress.

Sucrose synthase: expanding protein function.

SUCROSE SYNTHASE (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. This inference is based on the following observations: (a) tissue-specific, isoform-dependent and metabolically-regulated association of SUS with mitochondria and (b) isoform-specific and anoxia-responsive interaction of SUS with the voltage-dependent anion channel (VDAC), the major outer mitochondrial membrane protein. More recent work shows that both VDAC and SUS are also localized to the nucleus in maize seedling tissues. Their intricate regulation under anoxia indicates that these two proteins may have a role in inter-compartmental signaling.

Mitochondrial localization and putative signaling function of sucrose synthase in maize.

In many organisms, an increasing number of proteins seem to play two or more unrelated roles. Here we report that maize sucrose synthase (SUS) is distributed in organelles not involved in sucrose metabolism and may have novel roles beyond sucrose degradation. Bioinformatics analysis predicts that among the three maize SUS isoforms, SH1 protein has a putative mitochondrial targeting peptide (mTP). We validated this prediction by the immunodetection of SUS in mitochondria. Analysis with isoform-specific antisera revealed that both SH1 and SUS1 are represented in mitochondria, although the latter lacks a canonical mTP. The SUS2 isoform is not detectable in mitochondria, despite its presence in the cytosol. In maize primary roots, the mitochondrion-associated SUS (mtSUS; which includes SH1 and SUS1) is present mostly in the root tip, indicating tissue-specific regulation of SUS compartmentation. Unlike the glycolytic enzymes that occur attached to the outside of mitochondria, SH1 and SUS1 are intramitochondrial. The low abundance of SUS in mitochondria, its high Km value for sucrose, and the lack of sucrose in mitochondria suggest that mtSUS plays a non-sucrolytic role. Co-immunoprecipitation studies indicate that SUS interacts with the voltage-dependent anion channel in an isoform-specific and anoxia-enhanced manner and may be involved in the regulation of solute fluxes into and out of mitochondria. In several plant species, at least one of the SUS proteins possesses a putative mTP, indicating the conservation of the noncatalytic function across plant species. Taken together, these observations suggest that SUS has a novel noncatalytic function in plant cells.

Molecular and cellular adaptations of maize to flooding stress.

Anaerobic treatment dramatically alters the patterns of gene expression in maize (Zea mays L.) seedlings. During anaerobiosis there is an immediate repression of pre-existing protein synthesis, with the concurrent initiation of a selective synthesis of approx. 20 proteins. Among these anaerobic proteins are enzymes involved in glycolysis and related processes. However, inducible genes that have different functions were also found; these may function in other, perhaps more long-term, processes of adaptations to flooding, such as aerenchyma formation and root-tip death. In this article we review our recent work on maize responses to flooding stress, which has addressed two questions: how are these gene expression changes initiated and how do they lead to adaptation to flooding stress? Our results indicate that an early rise in cytosolic Ca(2+), as well as a quick establishment of ionic homeostasis, may be essential for the induction of adaptive changes at the cellular as well as organismal level.