Fibroblasts from wounds of different stages of repair vary in their ability to contract a collagen gel in response to growth factors

Wound contraction is one function of granulation tissue which is critical to repair. This study compares the ability of fibroblast-like cells derived from granulation tissue of various ages to contract a tissue equivalent, or a collagen gel, and examines the influence of growth factors implicated in wound repair on collagen gel contraction by these different cell populations. Cells from older granulation tissue (21 and 28 days) have an enhanced ability to contract a tissue equivalent when compared to cells from younger granulation tissue (7 and 14 days) or normal rat skin fibroblasts. Transforming growth factor-beta 1 (TGF-beta 1) enhanced contractility most in those cells which had a greater basal contractile ability. While basic fibroblast growth factor (bFGF) alone had moderately stimulatory effects at low doses (0.1-1.0 ng/ml), higher doses (greater than or equal to 10 ng/ml) inhibited basal contraction. Pretreatment with bFGF followed by exposure to TGF-beta 1, with or without the continued presence of bFGF, delayed gel contraction by cells from skin and early granulation tissue, but bFGF enhanced TGF-beta 1 activity in highly contractile cells. Transforming growth factor-alpha moderately enhanced contraction by cells from older granulation tissue. While both TGF-beta 1 and bFGF enhanced wound repair, their differential effects on the fibroblast-like cell derived from granulation tissue of different ages suggest that phenotypic differences exist between these cell populations. In addition, our results predict significant interactions between polypeptide cytokines at the site of repair.     

The indirect negative inotropic effects of the P1-receptor agonist, L-phenylisopropyladenosine, in guinea-pig isolated cardiac preparations: comparison with cromakalim.

The possible mechanisms of the indirect negative inotropic responses to the P1-receptor agonist, L-phenylisopropyladenosine (L-PIA) were evaluated in electrically paced (2 Hz, 5 ms pulse width, voltage 50% above threshold) left atria and papillary muscles of guinea pigs. The responses were compared in naive tissues (direct effects) or after prestimulation with submaximal concentrations of either cAMP-dependent positive inotropes (isoprenaline or forskolin) or the cAMP-independent inotrope Bay K 8644. Cumulative concentration-response curves were obtained in naive or prestimulated preparations for L-PIA or the potassium channel activator, cromakalim, for comparison. L-PIA and cromakalim exerted negative inotropy in naive atrial tissues, whereas only cromakalim was active in naive papillary muscles. In atria prestimulated with isoprenaline (31 nM) or forskolin (1.4 microM), the negative inotropy of L-PIA was enhanced compared with naive tissues. In contrast, prestimulation with Bay K 8644 (1 microM) exerted a significant functional antagonism of the response to L-PIA. In the case of cromakalim, prestimulation with isoprenaline exerted a functional antagonistic effect. In papillary muscles, an indirect negative inotropic effect of L-PIA was only seen in tissues prestimulated with the cAMP-dependent inotropes isoprenaline (31 nM) or forskolin (2.4 microM), and not in naive tissues or those prestimulated by Bay K 8644 (333 nM). As with atria, prestimulation with isoprenaline exerted a functional antagonistic effect on the response to cromakalim. These results suggest that the P1-receptor agonist, L-PIA, exerts its indirect negative inotropic effects in left atria by two mechanisms.2+ with cAMP-dependent positive inotropes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants

Our aim was to generate and prove the concept of "smart" plants to monitor plant phosphorus (P) status in Arabidopsis. Smart plants can be genetically engineered by transformation with a construct containing the promoter of a gene up-regulated specifically by P starvation in an accessible tissue upstream of a marker gene such as beta-glucuronidase (GUS). First, using microarrays, we identified genes whose expression changed more than 2.5-fold in shoots of plants growing hydroponically when P, but not N or K, was withheld from the nutrient solution. The transient changes in gene expression occurring immediately (4 h) after P withdrawal were highly variable, and many nonspecific, shock-induced genes were up-regulated during this period. However, two common putative cis-regulatory elements (a PHO-like element and a TATA box-like element) were present significantly more often in the promoters of genes whose expression increased 4 h after the withdrawal of P compared with their general occurrence in the promoters of all genes represented on the microarray. Surprisingly, the expression of only four genes differed between shoots of P-starved and -replete plants 28 h after P was withdrawn. This lull in differential gene expression preceded the differential expression of a new group of 61 genes 100 h after withdrawing P. A literature survey indicated that the expression of many of these "late" genes responded specifically to P starvation. Shoots had reduced P after 100 h, but growth was unaffected. The expression of SQD1, a gene involved in the synthesis of sulfolipids, responded specifically to P starvation and was increased 100 h after withdrawing P. Leaves of Arabidopsis bearing a SQD1::GUS construct showed increased GUS activity after P withdrawal, which was detectable before P starvation limited growth. Hence, smart plants can monitor plant P status. Transferring this technology to crops would allow precision management of P fertilization, thereby maintaining yields while reducing costs, conserving natural resources, and preventing pollution.     

Peripheral mitochondrial inner membrane protein, Mss2p, required for export of the mitochondrially coded Cox2p C tail in Saccharomyces cerevisiae

Cytochrome oxidase subunit 2 (Cox2p) is synthesized on the matrix side of the mitochondrial inner membrane, and its N- and C-terminal domains are exported across the inner membrane by distinct mechanisms. The Saccharomyces cerevisiae nuclear gene MSS2 was previously shown to be necessary for Cox2p accumulation. We have used pulse-labeling studies and the expression of the ARG8(m) reporter at the COX2 locus in an mss2 mutant to demonstrate that Mss2p is not required for Cox2p synthesis but rather for its accumulation. Mutational inactivation of the proteolytic function of the matrix-localized Yta10p (Afg3p) AAA-protease partially stabilizes Cox2p in an mss2 mutant but does not restore assembly of cytochrome oxidase. In the absence of Mss2p, the Cox2p N terminus is exported, but Cox2p C-terminal export and assembly of Cox2p into cytochrome oxidase is blocked. Epitope-tagged Mss2p is tightly, but peripherally, associated with the inner membrane and protected by it from externally added proteases. Taken together, these data indicate that Mss2p plays a role in recognizing the Cox2p C tail in the matrix and promoting its export.     

Zinc in plants

Zinc (Zn) is an essential component of thousands of proteins in plants, although it is toxic in excess. In this review, the dominant fluxes of Zn in the soil-root-shoot continuum are described, including Zn inputs to soils, the plant availability of soluble Zn(2+) at the root surface, and plant uptake and accumulation of Zn. Knowledge of these fluxes can inform agronomic and genetic strategies to address the widespread problem of Zn-limited crop growth. Substantial within-species genetic variation in Zn composition is being used to alleviate human dietary Zn deficiencies through biofortification. Intriguingly, a meta-analysis of data from an extensive literature survey indicates that a small proportion of the genetic variation in shoot Zn concentration can be attributed to evolutionary processes whose effects manifest above the family level. Remarkable insights into the evolutionary potential of plants to respond to elevated soil Zn have recently been made through detailed anatomical, physiological, chemical, genetic and molecular characterizations of the brassicaceous Zn hyperaccumulators Thlaspi caerulescens and Arabidopsis halleri.     

Evolutionary control of leaf element composition in plants

Leaf nitrogen (N) and phosphorus (P) concentrations are correlated in plants. Higher-level phylogenetic effects can influence leaf N and P. By contrast, little is known about the phylogenetic variation in the leaf accumulation of most other elements in plant tissues, including elements with quantitatively lesser roles in metabolism than N, and elements that are nonessential for plant growth. Here the leaf composition of 42 elements is reported from a statistically unstructured data set comprising over 2000 leaf samples, representing 670 species and 138 families of terrestrial plants. Over 25% of the total variation in leaf element composition could be assigned to the family level and above for 21 of these elements. The remaining variation corresponded to differences between species within families, to differences between sites which were likely to be caused by soil and climatic factors, and to variation caused by sampling techniques. While the majority of variation in leaf mineral composition is undoubtedly associated with nonevolutionary factors, identifying higher-level phylogenetic variation in leaf elemental composition increases our understanding of terrestrial nutrient cycles and the transfer of toxic elements from soils to living organisms. Identifying mechanisms by which different plant families control their leaf elemental concentration remains a challenge.     

Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea.A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS™ clones with insert sizes ∼20–40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs.Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter β-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue.This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor >40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.     

Gene expression changes in phosphorus deficient potato (Solanum tuberosum L.) leaves and the potential for diagnostic gene expression markers

Background

There are compelling economic and environmental reasons to reduce our reliance on inorganic phosphate (Pi) fertilisers. Better management of Pi fertiliser applications is one option to improve the efficiency of Pi fertiliser use, whilst maintaining crop yields. Application rates of Pi fertilisers are traditionally determined from analyses of soil or plant tissues. Alternatively, diagnostic genes with altered expression under Pi limiting conditions that suggest a physiological requirement for Pi fertilisation, could be used to manage Pifertiliser applications, and might be more precise than indirect measurements of soil or tissue samples.

Results

We grew potato (Solanum tuberosum L.) plants hydroponically, under glasshouse conditions, to control their nutrient status accurately. Samples of total leaf RNA taken periodically after Pi was removed from the nutrient solution were labelled and hybridised to potato oligonucleotide arrays. A total of 1,659 genes were significantly differentially expressed following Pi withdrawal. These included genes that encode proteins involved in lipid, protein, and carbohydrate metabolism, characteristic of Pi deficient leaves and included potential novel roles for genes encoding patatin like proteins in potatoes. The array data were analysed using a support vector machine algorithm to identify groups of genes that could predict the Pi status of the crop. These groups of diagnostic genes were tested using field grown potatoes that had either been fertilised or unfertilised. A group of 200 genes could correctly predict the Pi status of field grown potatoes.

Conclusions

This paper provides a proof-of-concept demonstration for using microarrays and class prediction tools to predict the Pi status of a field grown potato crop. There is potential to develop this technology for other biotic and abiotic stresses in field grown crops. Ultimately, a better understanding of crop stresses may improve our management of the crop, improving the sustainability of agriculture.     

Testing the distinctness of shoot ionomes of angiosperm families using the Rothamsted Park Grass Continuous Hay Experiment

? The ionome is the elemental composition of a tissue or organism. Phylogenetic variation in the ionomes of plant shoots has been widely reported based on controlled experiments, vegetation surveys and literature meta-analyses. However, environmental effects on phylogenetic variation in shoot ionomes have not been quantified. This study tests the hypothesis that phylogenetic variation in shoot ionomes is robust to environmental perturbation and that plant families can be distinguished by their shoot ionomes. ? Herbage was sampled from six subplots of the Rothamsted Park Grass Experiment. Subplots had received contrasting fertilizer treatments since 1856. Herbage was separated into its constituent species (n?=?21) and concentrations of eleven mineral elements were determined in dried shoot material. ? Shoot concentrations of calcium (Ca), zinc (Zn), manganese (Mn), magnesium (Mg) and sodium (Na) showed significant variation associated with plant species, and responded similarly to fertilizer treatments in diverse plant species. Species?×?treatment interactions were indicated for phosphorus (P), potassium (K), nickel (Ni), copper (Cu) and iron (Fe). Plant families could be distinguished by their shoot ionomes. The most informative elements for discriminant analysis were Ca?>?Mg?>?Ni?>?S?>?Na?>?Zn?>?K?>?Cu?>?Fe?>?Mn?>?P. ? Whilst shoot ionomes were sensitive to fertilizer treatment, phylogenetic variation in a subset of the shoot ionome (Ca, Zn, Mn, Mg) was robust to this environmental perturbation.