The Heat Stress Transcription Factor LlHsfA4 Enhanced Basic Thermotolerance through Regulating ROS Metabolism in Lilies (Lilium Longiflorum)

Heat stress severely affects the annual agricultural production. Heat stress transcription factors (HSFs) represent a critical regulatory juncture in the heat stress response (HSR) of plants. The HsfA1-dependent pathway has been explored well, but the regulatory mechanism of the HsfA1-independent pathway is still under-investigated. In the present research, HsfA4, an important gene of the HsfA1-independent pathway, was isolated from lilies (Lilium longiflorum) using the RACE method, which encodes 435 amino acids. LlHsfA4 contains a typical domain of HSFs and belongs to the HSF A4 family, according to homology comparisons and phylogenetic analysis. LlHsfA4 was mainly expressed in leaves and was induced by heat stress and H2O2 using qRT-PCR and GUS staining in transgenic Arabidopsis. LlHsfA4 had transactivation activity and was located in the nucleus and cytoplasm through a yeast one hybrid system and through transient expression in lily protoplasts. Over expressing LlHsfA4 in Arabidopsis enhanced its basic thermotolerance, but acquired thermotolerance was not achieved. Further research found that heat stress could increase H2O2 content in lily leaves and reduced H2O2 accumulation in transgenic plants, which was consistent with the up-regulation of HSR downstream genes such as Heat stress proteins (HSPs), Galactinol synthase1 (GolS1), WRKY DNA binding protein 30 (WRKY30), Zinc finger of Arabidopsis thaliana 6 (ZAT6) and the ROS-scavenging enzyme Ascorbate peroxidase 2 (APX2). In conclusion, these results indicate that LlHsfA4 plays important roles in heat stress response through regulating the ROS metabolism in lilies.

Mining Heat Stress Induced Genes Encoding Heat Shock Proteins (HSPs) and Heat Stress Transcription Factors (HSFs) in Strawberry (Fragaria x ananassa)

Plants have developed a variety of responses to extreme temperature situation in order to minimize damage and maintain cellular homeostasis. Recent research has revealed the involvement of heat stress proteins in thermotolerance in Arabidopsis. However, it is still obscure in strawberry plants. To identify heat stress responsive genes in strawberry (Fragaria x ananassa), we constructed a Transcriptome for strawberry and it was compared to Unigene annotated in several available proteomics, including Nr, COG, KEGC, and Swissport. Nineteen candidates with complete open reading frame (ORF) of 191 predicted genes encoding HSPs or HSFs were further analyzed. RT-PCR assays and heat stress induction analysis for these potential HSPs and HSFs were performed. Our data demonstrate that the prediction of potential heat stress responsive genes from our created Transcriptome for HSPs and HSFs, respectively, correlates well with the subsequent experimental results, indicating the Transcriptome we constructed could be translated into other scientific use.

Identification of biochemical differences between the sapwood and transition zone in Robinia pseudoacacia L. by differential display of proteins

The predominant proteins and enzymes in the sapwood and transition zones of Robinia pseudoacacia L. were identified and expressed by two methods: 2D SDS-PAGE (two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS). Large differences in the amount of proteins extracted were observed between the bark, sapwood and transition zones. Soluble proteins strongly expressed in sapwood have been identified, and the results interpreted to mean that these proteins are responsible for carbohydrate metabolism and flavonoid turnover. By contrast, proteins strongly expressed in the transition zone are mainly responsible for flavonoid biosynthesis. Lectins were found in protein fractions of both sapwood and the transition zone, and heat-stress proteins were detected only in the transition zone. The results are a further proof that flavonoids are synthesised directly at the transition zone between sapwood and heartwood, and that materials deposited in the sapwood are the source for synthesis of metabolites in heartwood, such as flavonoids and tannins.

Unexpected Stress-Reducing Effect of PhaP, a Poly(3-Hydroxybutyrate) Granule-Associated Protein, in Escherichia coli

ABSTRACTPhasins (PhaP) are proteins normally associated with granules of poly(3-hydroxybutyrate) (PHB), a biodegradable polymer accumulated by many bacteria as a reserve molecule. These proteins enhance growth and polymer production in natural and recombinant PHB producers. It has been shown that the production of PHB causes stress in recombinantEscherichia coli, revealed by an increase in the concentrations of several heat stress proteins. In this work, quantitative reverse transcription (qRT)-PCR analysis was used to study the effect of PHB accumulation, and that of PhaP fromAzotobactersp. strain FA8, on the expression of stress-related genes in PHB-producingE. coli. While PHB accumulation was found to increase the transcription ofdnaKandibpA, the expression of these genes and ofgroES,groEL,rpoH,dps, andyfiDwas reduced, when PhaP was coexpressed, to levels even lower than those detected in the non-PHB-accumulating control. These results demonstrated the protective role of PhaP in PHB-synthesizingE. coliand linked the effects of the protein to the expression of stress-related genes, especiallyibpA. The effect of PhaP was also analyzed in non-PHB-synthesizing strains, showing that expression of this heterologous protein has an unexpected protective effect inE. coli, under both normal and stress conditions, resulting in increased growth and higher resistance to both heat shock and superoxide stress by paraquat. In addition, PhaP expression was shown to reduce RpoH protein levels during heat shock, probably by reducing or titrating the levels of misfolded proteins.

The effect of chronic doxorubicin treatment on mitogen-activated protein kinases and heat stress proteins in rat hearts

The study has been designed to characterize protein systems involved in the responses of rat hearts to chronic doxorubicin (DOX) treatment. We investigated the influence of DOX on cardiac function, mitogen-activated protein kinases (MAPKs) and heat stress proteins (HSPs). Doxorubicin was administered to rats by intraperitoneal injections over a period of 6 weeks. In control and DOX-treated hearts exposed to 20 min global ischemia and 40 min reperfusion the recovery of contractile function after ischemia/reperfusion (I/R) was determined. The levels and phosphorylation state of proteins in tissue samples were analyzed using specific antibodies. We found an activation of extracellular signal-regulated kinases (ERKs) in rat hearts exposed to DOX treatment and better recovery of contractile function after I/R. Analysis of HSPs showed that DOX induced up-regulation of the levels of HSP60 and down-regulation of HSP70 levels. The levels and/or specific phosphorylation of other studied proteins (p38-MAPK, HSP27, HSP90) were not influenced by DOX. The results point to the possible role of ERKs and some HSPs in mechanisms underlying the response of rat hearts to chronic DOX treatment.