María Agustina Mazzella

AtTIP1;3 and AtTIP5;1, the only highly expressed Arabidopsis pollen‐specific aquaporins, transport water and urea

Pollination includes processes where water and/or solute movements must be finely regulated, suggesting participation of aquaporins. Using information available from different transcriptional profilings of Arabidopsis thaliana mature pollen, we showed that the only aquaporins that are selectively and highly expressed in mature pollen are two TIPs: AtTIP1;3 and AtTIP5;1. Pollen exhibited a lower number and more exclusive type of aquaporin expressed genes when compared to other single cell transcriptional profilings. When characterized using Xenopus oocyte swelling assays, AtTIP1;3 and AtTIP5;1 showed intermediate water permeabilities. Although they displayed neither glycerol nor boric acid permeability they both transported urea. In conclusion, these results suggest a function for AtTIP1;3 and AtTIP5;1 as specific water and urea channels in Arabidopsis pollen.

Temperature-dependent internode elongation in vegetative plants of Arabidopsis thaliana lacking phytochrome B and cryptochrome 1

Abstract. Vegetative plants of Arabidopsis thaliana (L.) Heynh. form a compact rosette of leaves in which internode growth is virtually arrested. Rapid extension of the internodes occurs after flower buds are present in the reproductive apex. Under natural radiation, continuous light from fluorescent lamps, or short photoperiods of light from fluorescent lamps, plants of the phyB cry1 double mutant (lacking both phytochrome B and cryptochrome 1) did not form normal rosettes because all the internodes showed some degree of elongation. Internode elongation was weak in the phyB single mutant and absent in the cry1 mutant, indicating redundancy between phytochrome B and cryptochrome 1. The absence of phytochrome A caused no effects. The failure to form normal rosettes was conditional because internode elongation was arrested at low temperatures in all the mutant combinations. In contrast, the temperature dependence of phytochrome B and cryptochrome 1 effects on hypocotyl growth was weak. The elongation of the internodes in phyB cry1 was not accompanied by early flowering as showed by the lack of effects on the final number of leaves. Apex dissection indicated that in phyB cry1 double mutants internode elongation anticipated the transition from the vegetative to the reproductive stage. Thus, stem growth in Arabidopsis thaliana is not fully dependent on the program of reproductive development.

Phytochrome A affects stem growth, anthocyanin synthesis, sucrose-phosphate-synthase activity and neighbour detection in sunlight-grown potato

Abstract. Phytochrome action in fully de-etiolated sunlight-grown potato (Solanum tuberosum L.) was studied by comparing wild-type (WT) plants and transgenic plants with either a sense or an anti-sense phytochrome A (phyA) construction. Radial stem growth, anthocyanin levels, and sucrose-phosphate-synthase activity were directly related to the levels of phyA (severely reduced in transgenics with anti-sense phyA, normal in WT and increased in transgenic with sense phyA). In contrast, longitudinal stem growth was inversely related to the levels of phyA. Phytochrome A influenced stem-extension growth responses to red/far-red ratios perceived by stable phytochrome[s]. First, far-red light reflected by non-shading neighbours promoted stem growth in WT plants but transgenic plants with either increased or reduced phyA levels failed to respond to this light signal. Second, plants with low phyA levels also showed impaired sensitivity to reductions in end-of-day red/far-red ratios. In addition, phyA appears to perceive changes in irradiance reaching the stem: lowering the amount of red plus far-red light reaching the stem promoted stem growth in WT plants. This effect was exaggerated in phyA overexpressors and absent in phyA underexpressors. Thus, phyA is active in fully de-etiolated, sunlight-grown plants.

Phytochrome Control of the Arabidopsis Transcriptome Anticipates Seedling Exposure to Light

Phytochromes mediate a profound developmental shift when dark-grown seedlings are exposed to light. Here, we show that a subset of genes is upregulated in phytochrome B (phyB) mutants even before dark-grown Arabidopsis thaliana seedlings are exposed to light. Most of these genes bear the RY cis motif, which is a binding site of the transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3), and the phyB mutation also enhances ABI3 expression. These changes in transcriptome have physiological consequences, because seedlings of the abi3 mutant showed enhanced responses to pulses of far-red light, whereas ABI3 overexpressers exhibited the opposite pattern. Seedlings of the wild type derived from seeds germinated in full darkness showed enhanced expression of genes bearing the RY cis motif and reduced responses to far-red light. We propose that, via changes in ABI3 expression, light, perceived mainly by phyB in the seed, generates a downstream transdevelopmental phase signal that preconditions the seedling to its most likely environment.

STIL, a peculiar molecule from styles, specifically dephosphorylates the pollen receptor kinase LePRK2 and stimulates pollen tube growth in vitro

BackgroundLePRK1 and LePRK2 are two pollen receptor kinases localized to the plasma membrane, where they are present in a high molecular weight complex (LePRK complex). LePRK2 is phosphorylated in mature and germinated pollen, but is dephosphorylated when pollen membranes are incubated with tomato or tobacco style extracts.ResultsHere we show that LePRK2 dephosphorylation is mediated by a heat-, acid-, base-, DTT- and protease-resistant component from tobacco styles. Using LePRK2 phosphorylation as a tracking assay for purification, style exudates were subjected to chloroform extraction, anionic exchange, and C18 reverse-phase chromatography columns. We finally obtained a single ~3,550 Da compound (as determined by UV-MALDI-TOF MS) that we named STIL (for Style Interactor for LePRKs). STIL increased pollen tube lengths of in vitro germinated pollen in a dose-dependent manner.ConclusionWe propose that the LePRK complex perceives STIL, resulting in LePRK2 dephosphorylation and an increase in pollen tube growth.

Hormonal networks involved in apical hook development in darkness and their response to light

In darkness, the dicot seedlings produce an apical hook as result of differential cell division and extension at opposite sides of the hypocotyl. This hook protects the apical meristem from mechanical damage during seedling emergence from the soil. In darkness, gibberellins act via the DELLA-PIF (PHYTOCHROME INTERACTING FACTORs) pathway, and ethylene acts via the EIN3/EIL1 (ETHYLENE INSENSITIVE 3/EIN3 like 1)-HLS1 (HOOKLESS 1) pathway to control the asymmetric accumulation of auxin required for apical hook formation and maintenance. These core pathways form a network with multiple points of connection. Light perception by phytochromes and cryptochromes reduces the activity of PIFs and (COP1) CONSTITUTIVE PHOTOMORPHOGENIC 1—both required for hook formation in darkness—, lowers the levels of gibberellins, and triggers hook opening as a component of the switch between heterotrophic and photoautotrophic development. Apical hook opening is thus a suitable model to study the convergence of endogenous and exogenous signals on the control of cell division and cell growth.

Finding unexpected patterns in microarray data.

We describe the performance of a protocol based on the sequential application of unsupervised and supervised methods to analyze microarray samples defined by a combination of factors. Correspondence analysis is used to visualize the emerging patterns of three set of novel or previously published data: photoreceptor mutants of Arabidopsis grown under different light/dark conditions, Arabidopsis exposed to different types of biotic and abiotic stress, and human acute leukemia. We find, for instance, that light has a dramatic effect on plants despite the absence of the four major photoreceptors, that bacterial-, fungal-, and viral-induced responses converge at later stages of attack, and that sample preparation procedures used in different hospitals have large effects on transcriptome patterns. We use canonical discriminant analysis to identify the genes associated with these patters and hierarchical clustering to find groups of coregulated genes that are easily visualized in a second round of correspondence analysis and ordered tables. The unconventional combination of standard descriptive multivariate methods offers a previously unrecognized tool to uncover unexpected information.

Metabolic responses to red/far-red ratio and ontogeny show poor correlation with the growth rate of sunflower stems

In sparse canopies, low red to far-red (R/FR) ratios reach only vertically-oriented stems, which respond with faster rates of extension. It is shown here that this signal also promotes stem dry matter accumulation in sunflower (Helianthus annuus) but not in mustard (Sinapis alba L.). Physically blocking internode extension growth also blocked internode recovery of labelled carbon fed to the leaves, indicating that increased carbon accumulation is partially a consequence of increased extension growth in sunflower. However, low R/FR also promoted carbon accumulation in the lower section of the internode, where extension growth was unaffected. Although the levels of many soluble metabolites and of cell-wall carbohydrates increased in the stem in response to low R/FR, allowing conservation of their concentration, sucrose was present at a lower concentration under low R/FR. This change is anticipated to favour carbon unloading from the stem phloem. Low R/FR also reduced the levels of selected fatty acids, fatty acid alcohols, and sterols. Compared with the lower section, the upper section of the internode showed higher levels of organic acids, amino acids, fatty acids, and sterols. It is concluded that the promotion of stem extension growth by low R/FR ratios causes increased dry matter gain in sunflower internodes by a mechanism that is largely independent of changes in metabolism, since, whilst both low R/FR and ontogeny alter the metabolic profile, the changes do not correlate with the observed growth responses.

Expression of stress-related proteins in Sediminibacterium sp. growing under planktonic conditions.

Aggregation is a common trait of bacteria in natural and engineered biological systems. Microbial aggregates, such as flocs, granules, and biofilms, are spatially heterogeneous environments. It is generally observed that by growing under aggregated conditions bacteria respond and adapt to environmental stress better than free‐swimming bacteria of the same species. We performed a proteomic analysis of a strain of Sediminibacterium, isolated from activated sludge, which grew planktonically in diluted culture media and in an aggregated form in media containing a high concentration of organic substrate. Auto‐aggregation was also observed in the presence of pyruvate in dilute media. Expression of a number of stress‐related proteins significantly increased under planktonic growth in comparison to aggregate growth. The upregulated proteins, identified by MALDI‐TOF mass spectrometry, were two isoforms of a protein belonging to the universal stress family (UspA), a thioredoxin‐disulfide reductase, the Campylobacter jejuni orthologue transcriptional regulator (Cj1172c), and the CocE/NonD hydrolase. We conclude that Sediminibaterium sp. C3 growth is stressed under planktonic conditions and that aggregation induced by pyruvate protects the bacteria against oxidative stress.

A proteome map of a quadruple photoreceptor mutant sustains its severe photosynthetic deficient phenotype.

Light is the environmental factor that most affects plant growth and development through its impact on photomorphogenesis and photosynthesis. A quadruple photoreceptor mutant lacking four of the most important photoreceptors in plants, phytochromes A and B (phyA, phyB) and cryptochromes 1 and 2 (cry1, cry2), is severely affected in terms of growth and development. Previous studies have suggested that in addition to a photomorphogenic disorder, the phyA phyB cry1 cry2 quadruple mutant might have severe alterations in photosynthetic ability. Here, we investigated the photosynthetic processes altered in the quadruple mutant and performed a proteomic profiling approach to identify some of the proteins involved. The phyA phyB cry1 cry2 quadruple mutant showed reduced leaf area and total chlorophyll content. Photosynthetic rates at high irradiances were reduced approximately 65% compared to the wild type (WT). Light-saturated photosynthesis and the response of net CO2 exchange to low and high internal CO2 concentrations suggest that the levels or activity of the components of the Calvin cycle and electron transport might be reduced in the quadruple mutant. Most of the under-expressed proteins in the phyA phyB cry1 cry2 quadruple mutant consistently showed a chloroplastic localization, whereas components of the Calvin cycle and light reaction centers were overrepresented. Additionally, Rubisco expression was reduced threefold in the phyA phyB cry1 cry2 quadruple mutant. Together, these results highlight the importance of the phytochrome and cryptochrome families in proper autotrophy establishment in plants. They also suggest that an overall limitation in the chlorophyll levels, expression of Rubisco, and enzymes of the Calvin Cycle and electron transport that affect ribulose-1,5-biphosphate (RuBP) regeneration reduced photosynthetic capacity in the phyA phyB cry1 cry2 quadruple mutant.