The distribution of NADPH-protochlorophyllide oxidoreductase in relation to chlorophyll accumulation along the barley leaf gradient

The possible regulatory role of NADPH-protochlorophyllide oxidoreductase for chlorophyll accumulation has been investigated in barley plants. Within the primary leaf of etiolated plants the different maturation stages of etioplasts are found in a linear series with the youngest in cells near the base and the oldest in cells near the tip. This distribution of different plastid forms is paralleled by drastic differences in the NADPH-protochlorophyllide-oxidoreductase content of the plastids and their capacity to accumulate chlorophyll during illumination. The amount of enzyme and the rate of chlorophyll accumulation are highest in the mature etioplast in the tip of the leaf and both decline rapidly with decreasing age of the leaf tissue, being almost undetectable in the leaf base. The translatable mRNA coding for the enzyme shows a different distribution pattern within the leaf. The highest concentration is found in the middle part of the leaf while in the top part only traces of this mRNA are detectable. It is concluded that during leaf development the enzyme is synthesized rapidly only during a limited time period and that it is stored subsequently in the mature etioplast as a stable protein. The close correlation between the distribution of the enzyme within the barley leaf and that of the potential to accumulate chlorophyll during illumination would favour a control of chlorophyll accumulation by the amount of NADPH-protochlorophyllide oxidoreductase. Dark-grown plants which were exposed to far-red light were used to test this possibility. The far-red-absorbing form of phytochrome (P_fr) has an inverse effect on the kinetics of chlorophyll accumulation and the enzyme concentration. Our results indicate that the rate of chlorophyll accumulation in barley is not determined by the level of NADPH-protochlorophyllide oxidoreductase present in the leaves.     

Study of the tumor microenvironment during breast cancer progression

Background

Different cells and mediators in the tumor microenvironment play important roles in the progression of breast cancer. The aim of this study was to determine the composition of the microenvironment during tumor progression in order to discover new related biomarkers and potentials for targeted therapy.

Methods

In this study, breast cancer biopsies from four different stages, and control breast biopsies were collected. Then, the mRNA expression of several markers related to different CD4⁺ T cell subsets including regulatory T cells (Treg), T helper (Th) type 1, 2 and 17 were determined. In addition, we investigated the expression of two inflammatory cytokines (TNF-α and IL-6) and inflammatory mediators including FASL, IDO, SOCS1, VEGF, and CCR7.

Results

The results showed that the expression of Th1 and Th17 genes was decreased in tumor tissues compared to control tissues. In addition, we found that the gene expression related to these two cell subsets decreased during cancer progression. Moreover, the expression level of TNF-α increased with tumor progression.

Conclusion

We conclude that the expression of genes related to immune response and inflammation is different between tumor tissues and control tissues. In addition, this difference was perpetuated through the different stages of cancer.

     

KAS IV: a 3-ketoacyl-ACP synthase from Cuphea sp . is a medium chain specific condensing enzyme

cDNA clones encoding a novel 3-ketoacyl-ACP synthase (KAS) have been isolated from Cuphea . The amino acid sequence of this enzyme is different from the previously characterized classes of KASs, designated KAS I and III, and similar to those designated as KAS II. To define the acyl chain specificity of this enzyme, we generated transgenic Brassica plants over-expressing the cDNA encoded protein in a seed specific manner. Expression of this enzyme in transgenic Brassica seeds which normally do not produce medium chain fatty acids does not result in any detectable modification of the fatty acid profile. However, co-expression of the Cuphea KAS with medium chain specific thioesterases, capable of production of either 12:0 or 8:0/10:0 fatty acids in seed oil, strongly enhances the levels of these medium chain fatty acids as compared with seed oil of plants expressing the thioesterases alone. By contrast, co-expression of the Cuphea KAS along with an 18:0/18:1-ACP thioesterase does not result in any detectable modification of the fatty acids. These data indicate that the Cuphea KAS reported here has a different acyl-chain specificity to the previously characterized KAS I, II and III. Therefore, we designate this enzyme KAS IV, a medium chain specific condensing enzyme.     

Functional Convergence of Oxylipin and Abscisic Acid Pathways Controls Stomatal Closure in Response to Drought

Membranes are primary sites of perception of environmental stimuli. Polyunsaturated fatty acids are major structural constituents of membranes that also function as modulators of a multitude of signal transduction pathways evoked by environmental stimuli. Different stresses induce production of a distinct blend of oxygenated polyunsaturated fatty acids, “oxylipins.” We employed three Arabidopsis (Arabidopsis thaliana) ecotypes to examine the oxylipin signature in response to specific stresses and determined that wounding and drought differentially alter oxylipin profiles, particularly the allene oxide synthase branch of the oxylipin pathway, responsible for production of jasmonic acid (JA) and its precursor 12-oxo-phytodienoic acid (12-OPDA). Specifically, wounding induced both 12-OPDA and JA levels, whereas drought induced only the precursor 12-OPDA. Levels of the classical stress phytohormone abscisic acid (ABA) were also mainly enhanced by drought and little by wounding. To explore the role of 12-OPDA in plant drought responses, we generated a range of transgenic lines and exploited the existing mutant plants that differ in their levels of stress-inducible 12-OPDA but display similar ABA levels. The plants producing higher 12-OPDA levels exhibited enhanced drought tolerance and reduced stomatal aperture. Furthermore, exogenously applied ABA and 12-OPDA, individually or combined, promote stomatal closure of ABA and allene oxide synthase biosynthetic mutants, albeit most effectively when combined. Using tomato (Solanum lycopersicum) and Brassica napus verified the potency of this combination in inducing stomatal closure in plants other than Arabidopsis. These data have identified drought as a stress signal that uncouples the conversion of 12-OPDA to JA and have revealed 12-OPDA as a drought-responsive regulator of stomatal closure functioning most effectively together with ABA.To colonize a diverse range of environments successfully, plants have developed converging functional pathways to synthesize an array of secondary metabolites for their protection against hostile conditions. For example, in response to environmental challenges, the oxylipin pathway induces the de novo synthesis of biologically active compounds called “oxylipins,” derivatives of oxygenated polyunsaturated fatty acids (Feussner and Wasternack, 2002; Howe and Schilmiller, 2002). Among the oxylipin pathways, the enzymes allene oxide synthase (AOS) and hydroperoxide lyase (HPL) are considered to partition two major branches that compete for the same substrates and are critical plant stress response pathways (Chehab et al., 2008).Production of the AOS pathway metabolites 12-oxo-phytodienoic acid (12-OPDA) and jasmonic acid (JA) originates from α-linolenic acid of chloroplast membranes (Feussner and Wasternack, 2002). Oxygenation of α-linolenic acid by a 13-lipoxygenase followed by the action of AOS forms an unstable allene oxide that is subsequently cyclized by an allene oxide cyclase to form 12-OPDA (Stenzel et al., 2012). 12-OPDA is the end product of the plastid-localized part of the pathway (Stintzi and Browse, 2000; Schaller and Stintzi, 2009). 12-OPDA is then translocated to the peroxisome where it is reduced by 12-OPDA reductase3 (OPR3) and subsequently activated by CoA ester prior to undergoing three rounds of β-oxidation to form JA (Schaller et al., 2000; Koo et al., 2006; Kienow et al., 2008). 12-OPDA is also a signaling molecule with both overlapping and distinct functions from JA. The Arabidopsis (Arabidopsis thaliana) opr3 mutant is deficient in JA synthesis but accumulates 12-OPDA and displays wild-type resistance to the dipteran Bradysia impatiens and to the fungal pathogen Alternaria brassicicola, generally considered JA-dependent responses (Stintzi et al., 2001). In addition, expression studies have identified genes induced by 12-OPDA but not by JA or methyl jasmonate (MeJA; Kramell et al., 2000; Stintzi et al., 2001; Taki et al., 2005; Ribot et al., 2008). These studies collectively show that 12-OPDA mediates gene expression with or without the canonical JA signaling framework (Stintzi et al., 2001; Taki et al., 2005; Ribot et al., 2008).The HPL branch of the oxylipin pathway produces aldehydes and corresponding alcohols. The first enzyme in the pathway is encoded by one or more HPL genes, differing in their subcellular localization, including microsomes (Pérez et al., 1999), lipid bodies (Mita et al., 2005), and the outer envelope of chloroplasts (Froehlich et al., 2001), and in some cases, with no specific localization in a particular organelle (Noordermeer et al., 2000). This variation in the number of genes and subcellular localization of their encoded enzymes is suggestive of the differential regulation of this pathway and, ultimately, the diversity of their responses, potentially tailored to the nature of stimuli.We have previously identified three rice (Oryza sativa) HPLs (HPL1 through HPL3) differing in their enzyme kinetics and substrate preference. Expression of these enzymes in Arabidopsis accession Columbia (Col-0), a natural hpl loss-of-function mutant, reestablished the production of the pathway metabolites (Chehab et al., 2006) and revealed the key role of HPL-derived metabolites in plant stress signaling (Chehab et al., 2008).The HPL and AOS branches of the oxylipin pathway do not function independently; the signaling crosstalk between them is key to fine tuning plant adaptive responses to a diverse range of perturbations (Halitschke et al., 2004; Liu et al., 2012; Scala et al., 2013).To gain deeper insight into the role of AOS- and HPL-derived metabolites in fine-tuning plant stress responses, we have (1) characterized the corresponding oxylipin signatures in response to wounding and drought in three Arabidopsis ecotypes, (2) generated a range of transgenic lines that produce varying blends of oxylipins tailored to the nature of the stress, (3) elucidated a JA-independent role for 12-OPDA in enhanced drought tolerance in part via regulation of stomatal aperture, and (4) reexamined the 12-OPDA-mediated regulation of stomatal aperture, alone or in combination with abscisic acid (ABA) in the model system Arabidopsis as well as in two crop species, namely tomato (Solanum lycopersicum) and Brassica napus. Unexpectedly, these analyses have identified drought as a stress signal that uncouples the conversion of 12-OPDA to JA and have revealed that 12-OPDA is a previously unrecognized regulator of stomatal closure in response to drought. This function of 12-OPDA, however, is most effective when combined with ABA, a phytohormone known to be essential for plant-adaptive responses to drought stress (Seki et al., 2007).     

The function of proteases during the light-dependent transformation of etioplasts to chloroplasts in barley (Hordeum vulgare L.)

The role of proteolysis during the light-induced rapid decrease of the NADPH: protochlorophyllide oxidoreductase in barley was studied. A proteolytic activity with a pH optimum of 4.5 was present in a plastid preparation of etiolated barley seedlings. No other proteolytic activity could be detected. The temperature optimum for the proteolysis was 50°C, and the highest specific activity was measured with hemoglobin as the substrate. In contrast to previous proposals, no evidence for the specific involvement of this protease was found during the light-induced transformation of etioplasts to chloroplasts.