On the Inside

Peter V. Minorsky

doi:10.1104/pp.104.900212

Published December 2006. DOI: https://doi.org/10.1104/pp.104.900212

Pollen Tip Growth and Oscillations in NAD(P)H

The growth rates of lily (Lilium formosanum) pollen tubes oscillate with a period of 20 to 50 s during polarized tip growth. Many physiological processes underlying polarized tip growth also oscillate with the same period as growth but not with the same phase and amplitude. Among the oscillatory processes, most attention has been directed toward ions. Cl⁻ efflux mirrors growth rate, whereas changes in intracellular and extracellular Ca²⁺, as well as extracellular H⁺ and K⁺, occur after growth. In searching for events that anticipate and potentially lead growth, changes in metabolism are of special interest since energy production and the processes that drive the synthesis of new cell wall material must at some level precede and anticipate the changes in growth rate. The high energy status and reducing power of NADH and NADPH collectively [NAD(P)H] drive many key biosynthetic reactions and ATP production. Moreover, since NAD(P)H but not NAD(P)⁺ possesses an endogenous fluorescence, it is possible to detect the reduced form in living cells. Cárdenas et al. (pp. 1460–1468) have examined changes in NAD(P)H during oscillatory pollen tube growth. They report that NAD(P)H oscillates with the same period as growth but not the same phase. The strongest signal resides 20 to 40 μm behind the apex where mitochondria accumulate. The authors suggest that the changes in fluorescence reflect an oscillation between the reduced (peaks) and oxidized (troughs) states of NAD(P)H, and that an increase in the oxidized state [NAD(P)⁺] may be coupled to the synthesis of ATP. The troughs in NAD(P)H appear to anticipate changes in growth rate and, thus, might be key players in polarized tip growth.

Ethylene and Nutations

Nutations are oscillatory movements caused by localized differential growth in plant organs. In the course of examining the effects of ethylene on the kinetics of apical hook closure and hypocotyl thickening in Arabidopsis (Arabidopsis thaliana) seedlings, Binder et al. (pp. 1690–1700) made some serendipitous discoveries concerning nutations and ethylene. Using time-lapse imaging to examine the long-term effects of ethylene on etiolated Arabidopsis seedlings, the authors discovered that ethylene stimulates nutations of the hypocotyls following an average delay of 6 h. Ethylene responses in Arabidopsis are mediated by a family of five receptors (ETR1, ERS1, ETR2, EIN4, and ERS2). Ethylene-stimulated nutations were eliminated in etr1-7 loss-of-function mutants. Transformation of the etr1-7 mutant with a wild-type genomic ETR1 transgene rescued the nutation phenotype, suggesting that ETR1 is required for nutations. Loss-of-function mutations in the other receptor isoforms had no effect on ethylene-stimulated nutations. Surprisingly, the double ers1-2 ers2-3 and triple etr2-3 ers2-3 ein4-4 loss-of-function mutants constitutively nutated in air. These results support a model where all the receptors are involved in ethylene-stimulated nutations. The ETR1 receptor, however, is required and has a contrasting role from the other receptor isoforms in regulating nutation.

A Negative Regulator of ABA Function

Abscisic acid (ABA) signaling networks in plants have proven to be quite complex, and the contribution by Kariola et al. (pp. 1559–1573) suggests that they are more complex still. The authors report that EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15), a small, acidic protein with no known function, is a key negative regulator of ABA responses in plants. Although ERD15 was recognized as a rapidly drought-responsive gene in Arabidopsis, the authors show that alterations of ERD15 expression modulate ABA responsiveness in Arabidopsis. Overexpression of ERD15 reduced the ABA sensitivity of Arabidopsis as manifested in decreased drought tolerance and in impaired ability of the plants to increase their freezing tolerance in response to this hormone. In contrast, RNAi silencing of ERD15 resulted in plants that were hypersensitive to ABA and showed improved tolerance to both drought and freezing, as well as impaired seed germination, in the presence of ABA. The authors also show that ERD15 is induced by pathogen attack and that overexpression of this gene enhances salicylic acid-dependent pathogen defense and plant resistance to the bacterial necrotroph Erwinia carotovora. Thus, ERD15 is a novel mediator of stress-related ABA signaling in Arabidopsis and may play an important role in mediating cross talk between biotic and abiotic stress responses.

Reversibly Glycosylated Polypeptides and Pollen Development

Because reversibly glycosylated polypeptides (RGPs) colocalize with the Golgi apparatus and are able to react with UDP-sugars, they have been implicated in polysaccharide biosynthesis. The Arabidopsis genome contains five RGP genes. Drakakaki et al. (pp. 1480–1492) have characterized the native expression pattern of Arabidopsis RGP1 and RGP2 and used reverse genetics to investigate their respective functions. Although both genes are expressed ubiquitously, the highest levels are observed in actively growing tissues, especially in mature pollen. Single-gene disruptions did not show any obvious morphological defects, whereas the double mutant is lethally affected. Detailed analyses revealed that mutant pollen development is associated with abnormally enlarged vacuoles and a poorly defined inner cell wall layer, which consequently results in disintegration of the pollen structure during the first mitotic division of pollen. These results suggest that RGP1 and RGP2 are required during microspore development and pollen mitosis, and that they may affect cell division, vacuolar integrity, or both.

Natural Variation under Carbon-Limiting Conditions

Plant growth is fueled by the photosynthetic assimilation of carbon (C) and the assimilation of inorganic nutrients, of which nitrogen (N) is quantitatively the most important. Studies of natural variations in accessions can be used to uncover correlations between parameters across large sets of genotypes, which may provide insights into the structure of physiological, metabolic, or regulatory networks. Cross et al. (pp. 1574–1588) have investigated which metabolic parameters vary and which parameters change in a coordinated manner in 24 genetically diverse Arabidopsis accessions, grown under C-limited conditions. This research was facilitated by recently established sensitive microplate-based assays, which allow rapid measurements of metabolites and enzyme activities in C and N metabolism. The 24 accessions were grown in short days, moderate light, and high nitrate, and analyzed for rosette biomass, levels of structural components (protein, chlorophyll), total phenols and major metabolic intermediates (sugars, starch, nitrate, amino acids), and the activities of seven representative enzymes that are central to C and N metabolism. Rosette size tended to be positively correlated with higher activities of enzymes in central C and N metabolism, and relatively unaffected by levels of key C and N metabolites. Therefore, the authors conclude that growth is not related to the absolute levels of starch, sugars, and amino acids; instead, it is related to flux, which is indicated by the enzymatic capacity to use these central resources.

Proteomics of Seed Dormancy

The Arabidopsis accession from the Cape Verde Islands (Cvi) displays a much deeper seed dormancy than the two accessions that are most widely studied (Columbia and Landsberg erecta). Chibani et al. (pp. 1493–1510) have carried out a proteomic analysis of seed dormancy in Cvi in dormant, freshly harvested seeds, and non-dormant, after-ripened seeds. Their results suggest that proteins associated with metabolic functions accumulate during after-ripening in the dry state leading to dormancy release. Exogenous application of ABA to non-dormant seeds strongly impeded germination. This raises the question of whether natural seed dormancy is physiologically identical to ABA-induced dormancy. The authors report that the protein profiles of dormant seeds were markedly different from those obtained with the non-dormant, after-ripened seeds imbibed in ABA. Thus, the mechanisms blocking germination of the non-dormant seeds by ABA application appear to be different from those preventing germination of naturally dormant seeds.

Transcriptomics of the Vasculature of Plantago major Leaf Blades

Pure, intact vascular tissue can be isolated easily and rapidly from the leaf blades of Plantago major (common plantain), a plant model often used for molecular studies of phloem transport. Pommerrenig et al. (pp. 1427–1441) present data concerning the generation, characterization, and application of tools for the analysis of vasculature-specific gene expression in Plantago leaf blades. More than 3,200 independent mRNAs were identified. The specificity of the library was confirmed by the identification of expressed sequence tags (ESTs) of genes previously identified in Plantago companion cells and by the high expression levels of genes known to be vascular-specific in other plants. The authors also identified pathways and enzymes that hitherto have not been widely recognized as being vasculature specific or vasculature typical. For example, they identified mRNAs that encode a mannitol dehydrogenase, an α-amylase, and a Suc phosphate synthase, as well as several mRNAs for the polyamine biosynthetic pathway. The authors also established a transformation technique that they successfully used to confirm the vascular specificity of a Plantago promoter/β-glucuronidase construct. The applicability of the obtained data was also demonstrated for other plant species. Reporter gene constructs generated with promoters from Arabidopsis homologs of newly identified Plantago vascular ESTs revealed vascular specificity of these genes in Arabidopsis also. The data presented concerning vascular ESTs and the newly developed transformation system represent important tools for future studies of the functional genomics of the vasculature of plants.