The effect of 15 consecutive days of heat–exercise acclimation on heat shock protein 70

The purpose of this study was to investigate the alterations in serum heat shock protein (Hsp) 70 levels during a 15-consecutive-day intermittent heat–exercise protocol in a 29-year-old male ultra marathon runner. Heat acclimation, for the purpose of physical activities in elevated ambient temperatures, has numerous physiological benefits including mechanisms such as improved cardiac output, increased plasma volume and a decreased core temperature (T _c). In addition to the central adaptations, the role of Hsp during heat acclimation has received an increasing amount of attention. The acclimation protocol applied was designed to correspond with the athlete’s tapering period for the 2007 Marathon Des Sables. The subject (VO₂max = 50.7 ml·kg⁻¹·min⁻¹, peak power output [PPO] = 376 W) cycled daily for 90 min at a workload corresponding to 50% of VO₂max in a temperature-controlled room (average WBGT = 31.9 ± 0.9°C). Venous blood was sampled before and after each session for measurement of serum osmolality and serum Hsp70. In addition, T _c, heart rate (HR) and power output (PO) was measured throughout the 90 min to ensure that heat acclimation was achieved during the 15-day period. The results show that the subject was successfully heat acclimated as seen by the lowered HR at rest and during exercise, decreased resting and exercising T _c and an increased PO. The heat exercise resulted in an initial increase in Hsp70 concentrations, known as thermotolerance, and the increase in Hsp70 after exercise was inversely correlated to the resting values of Hsp70 (Spearman’s rank correlation = −0.81, p < 0.01). Furthermore, the 15-day heat–exercise protocol also increased the basal levels of Hsp70, a response different from that of thermotolerance. This is, as far as we are aware, the first report showing Hsp70 levels during consecutive days of intermittent heat exposure giving rise to heat acclimation. In conclusion, a relatively longer heat acclimation protocol is suggested to obtain maximum benefit of heat acclimation inclusive of both cellular and systemic adaptations.     

Effect of Methyl Jasmonate on Morphology and Dormancy Development in Lily Bulblets Regenerated In Vitro

Scales of lily bulbs are swollen petioles. Lily scale fragments cultured in vitro regenerate bulblets consisting of scales that may or may not carry a leaf blade. The bulblets are dormant and require a cold treatment to sprout. We added the gaseous plant growth regulator methyl jasmonic acid (MeJA) in the headspace of the tissue-culture container and studied the effect on plantlet morphology (scale/leaf-blade formation) and dormancy development in three lilies, Lilium speciosum “Rubrum No. 10,” L. longiflorum “Snow Queen,” and the Asiatic hybrid “Connecticut King.” Methyl jasmonic acid strongly reduced leaf-blade formation in Lilium longiflorum and Connecticut King. This was a specific effect as scale formation was affected much less. The specific inhibition of leaf-blade formation was not observed in Lilium speciosum. In this lily, high concentrations of methyl jasmonic acid (MeJA) inhibited leaf-blade and scale formation to similar extents. Methyl jasmonic acid reduced dormancy development in all three lilies, with the largest effect observed in Connecticut King. In this Asiatic hybrid, almost all bulblets that had regenerated at 300 or 1000 μl l⁻¹ MeJA in the headspace, did not require a dormancy-breaking treatment to achieve sprouting after planting in soil. Previously, it has been found in lily that treatments that reduce leaf-blade formation promote dormancy development. The present findings with MeJA do not agree with this. In the three lilies, the various parameters that were studied—regeneration, scale weight, leaf-blade weight, and dormancy development—were very differently affected by MeJA.     

Heat stress-induced BBX18 negatively regulates the thermotolerance in Arabidopsis

There is increasing evidence for considerable interlinking between the responses to heat stress (HS) and light signaling. In the present work, we provide molecular evidence that BBX18, a negative regulator in photomorphogenesis belonging to the B-box zinc finger protein family in Arabidopsis thaliana, is involved in the regulation of thermotolerance. Using quantitative RT-PCR, GUS staining and immunoblot analysis, our results indicate that the expression of BBX18 was induced by HS. BBX18-RNAi and 35S::BBX18 transgenic Arabidopsis plants were obtained for functional analysis of BBX18. Under-expression of BBX18 displayed increased both basal and acquired thermotolerance in the transgenic plants, while over-expression of BBX18 reduced tolerance to HS in transgenic lines. Moreover, when wild-type, BBX18-RNAi and 35S::BBX18 transgenic plants were treated with HS, HR-related digalactosyldiacylglycerol synthase 1 (DGD1) was down-regulated by BBX18 in both normal and heat shock conditions. Besides, the expression levels of Hsp70, Hsp101 and APX2 were increased in BBX18-RNAi transgenic plants, but lower in 35S::BBX18 transgenic plants. However, the expression of HsfA2 was lower in BBX18-RNAi transgenic plants and higher in the 35S::BBX18 after high-temperature treatment. These results suggesting that, by modulated expression of a set of HS-responsive genes, BBX18 weakened tolerance to HS in Arabidopsis. So our data indicate that BBX18 plays a negative role in thermotolerance.     

Heat-tolerant basmati rice engineered by over-expression of hsp101

Rice is sensitive to high-temperature stress at almost all the stages of its growth and development. Considering the crucial role of heat shock protein 101 (Hsp101) in imparting thermotolerance to cells, we introduced Arabidopsis thaliana hsp101 (Athsp101) cDNA into the Pusa basmati 1 cultivar of rice (Oryza sativa L.) by Agrobacterium-mediated transformation. Stable integration and expression of the transgene into the rice genome was demonstrated by Southern, northern and western blot analyses. There appeared no adverse effect of over-expression of the transgene on overall growth and development of transformants. The genetic analysis of tested T₁ lines showed that the transgene segregated in a Mendelian fashion. We compared the survival of T₂ transgenic lines after exposure to different levels of high-temperature stress with the untransformed control plants. The transgenic rice lines showed significantly better growth performance in the recovery phase following the stress. This thermotolerance advantage appeared to be solely due to over-expression of Hsp101 as neither the expression of low-molecular-weight heat shock proteins (HSPs) nor of other members of Clp family proteins was altered in the transgenic rice. The production of high temperature tolerant transgenic rice cultivars would provide a stability advantage under supra-optimal temperature regime thereby improving its overall performance.     

Photosynthetic Thermotolerance is Quantitatively and Positively Correlated with Production of Specific Heat-shock Proteins Among Nine Genotypes of Lycopersicon (Tomato)

We recently showed that the chloroplast small heat-shock protein (herein referred to as chlp Hsp24) protects photosystem 2 (PS2) during heat stress, and phenotypic variation in production of chlp Hsp24 is positively related to PS2 thermotolerance. However, the importance of chlp Hsp24 or other Hsps to other aspects of photosynthesis and overall photosynthetic thermotolerance is unknown. To begin investigating this and the importance of genetic variation in Hsp production to photosynthetic thermotolerance, the production of several prominent Hsps and photosynthetic thermotolerance were quantified in nine genotypes of Lycopersicon, and then the relationships between thermotolerance of net photosynthetic rate (P _N) and production of each Hsp were examined. The nine genotypes exhibited wide variation in P _N thermotolerance and production of each of the Hsps examined (chlp Hsp70, Hsp60, and Hsp24, and cytosol Hsp70). No statistically significant relationship was observed between production of chlp Hsp70 and P _N thermotolerance, and only a weak positive relationship between cytosolic Hsp70 and P _N was detected. However, significant positive relationships were observed between production of chlp Hsp24 and Hsp60 and P _N thermotolerance. Hence natural variation in production of chlp Hsp24 and Hsp60 is important in determining variation in photosynthetic thermotolerance. This is perhaps the first evidence that chlp Hsp60 is involved in photosynthetic thermotolerance, and these in vivo results are consistent with previous in vitro results showing that chlp Hsp24 protects PS2 during heat stress.     

Expression and functional analysis of two genes encoding transcription factors, VpWRKY1 and VpWRKY2, isolated from Chinese wild Vitis pseudoreticulata

In this study, two WRKY genes were isolated from Erysiphe necator-resistant Chinese wild Vitis pseudoreticulata W. T. Wang ‘Baihe-35-1’, and designated as VpWRKY1 (GenBank accession no. GQ884198) and VpWRKY2 (GenBank accession no. GU565706). Nuclear localization of the two proteins was demonstrated in onion epidermal cells, while trans-activation function was confirmed in the leaves of ‘Baihe-35-1’. Expression of VpWRKY1 and VpWRKY2 was induced rapidly by salicylic acid treatment in ‘Baihe-35-1’. Expression of VpWRKY1 and VpWRKY2 was also induced rapidly by E. necator infection in 11 grapevine genotypes; the maximum induction of VpWRKY1 was greater in E. necator-resistant grapevine genotypes than in susceptible ones post E. necator inoculation. Furthermore, ectopic expression of VpWRKY1 or VpWRKY2 in Arabidopsis enhanced resistance to powdery mildew Erysiphe cichoracearum, and enhanced salt tolerance of transgenic plants. VpWRKY2 also enhanced cold tolerance of transgenic plants. In addition, the two proteins were shown to regulate the expression of some defense marker genes in Arabidopsis and grapevine. The data suggest that VpWRKY1 and VpWRKY2 may underlie the resistance in transgenic grapevine to E. necator and tolerance to salt and cold stresses.     

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H⁺ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 μM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H⁺ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H⁺ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen. Received: 20 June 2000 / Accepted: 2 October 2000     

Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease

 A 1.1-kb DNA fragment containing the coding region of a thaumatin-like protein (TLP-D34), a member of the PR-5 group, was cloned into the rice transformation vector pGL2, under the control of the CaMV 35S promoter. The Indica rice cultivars, ‘Chinsurah Boro II’, ‘IR72’, and ‘IR51500’ were transformed with the tlp gene construct by PEG-mediated direct gene transfer to protoplasts and by biolistic transformation using immature embryos. The presence of the chimeric gene in T₀, T₁, and T₂ transgenic plants was detected by Southern blot analysis. The presence of the expected 23-kDa TLP in transgenic plants was confirmed by Western blot analysis and by staining with Coomassie Brilliant Blue. Bioassays of transgenic plants challenged with the sheath blight pathogen, Rhizoctonia solani, indicated that over-expression of TLP resulted in enhanced resistance compared to control plants. Received: 11 August 1998 / Accepted: 26 August 1998     

Transformation of a muskmelon ‘Galia’ hybrid parental line (Cucumis melo L. var. reticulatus Ser.) with an antisense ACC oxidase gene

‘Galia’ muskmelon (Cucumis melo L. var. reticulatus Ser.) has been recalcitrant to transformation by Agrobacterium tumefaciens. Transformation of the ‘Galia’ male parental line, ‘Krymka’, with an ACC oxidase (CMACO-1) gene in antisense orientation is described herein. Explants were transformed using A. tumefaciens strain ABI, which contained a vector pCmACO1-AS plasmid, bearing an antisense gene of CMACO-1 and the CP4 syn gene (glyphosate-tolerance). Both CMACO-1 and CP4 syn genes were assessed by a polymerase chain reaction method. Flow cytometry analysis was performed to determine plant ploidy level of primary transformants. Two completely diploid independent transgenic plants were obtained. Southern blot and segregation analysis in the T₁ generation determined that each independent transgenic line had one single insertion of the transgene. These transgenic muskmelon male parental lines have potential for use in the production of ‘Galia’ F₁ hybrids with improved shelf life.