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Broad-Complex (BrC) is a downstream target of both 20-hydroxyecdysone and juvenile hormone signalling. BrC regulates morphogenetic changes between nymphal instars in hemimetabolans, whereas it controls pupal commitment, pupal morphogenesis and inhibits adult differentiation in holometabolans. Among five BrC cDNAs (Z1-Z4 and Z6) identified in the Colorado potato beetle, we found in this work that Z1, Z2 and Z6 were mainly expressed at the last (fourth) instar and prepupal stages, whereas the levels of Z3 and Z4 increased during the penultimate (third) instar stage, peaked at the last instar larval phase and gradually decreased at the prepupal and pupal periods. When knocking down all BrC isoforms by RNA interference (RNAi) at the penultimate instar stage, around 20% of the resultant larvae remained as moribund beetles. These moribund BrC RNAi larvae were completely or partially wrapped in old cuticle. Likewise, a portion of larvae treated for a single double-stranded RNA of Z3, Z4 or Z6 displayed a degree of similar aberrancies, increasing in the order of isoforms Z6

Two Drosophila melanogaster E-twenty-six domain transcription factor isoforms (E74A and E74B) act differentially at the start of the 20-hydroxyecdysone (20E) signalling cascade to regulate larval-pupal metamorphosis. In the present paper, we identified the two isoforms (LdE74A and LdE74B) in Leptinotarsa decemlineata. During the larval development stage, the mRNA transcript levels of the two LdE74 isoforms were correlated with circulating 20E titres. In vitro midgut culture and in vivo dietary supplementation with 20E revealed that the presence of 20E induced expression peaks of both LdE74A and LdE74B, with similar patterns observed for the two isoforms. Moreover, the mRNA transcript levels of both LdE74A and LdE74B isoforms were significantly downregulated in the L. decemlineata ecdysone receptor RNA interference (RNAi) specimens, but not in the LdE75 RNAi beetles. Ingestion of 20E reduced the larval fresh weights and shortened the larval development period, irrespective of knockdown of LdE74 or not. RNAi of LdE74 did not affect 20E-induced expression of the Ecdysone induced protein 75-hormone receptor 3-fushi tarazu factor 1 (E75-HR3-FTZ-F1) transcriptional cascade. Thus, it seems that LdE74 mediates 20E signalling independent of the E75-HR3-FTZ-F1 transcriptional cascade. Furthermore, silencing of both LdE74 isoforms caused failure of ecdysis. Most of the LdE74 RNAi beetles remained as prepupae. The LdE74 RNAi prepupae exhibited adult character-like forms underneath after removal of the apolysed larval cuticle. Their appendages such as antennae, legs and wings were shorter than those of control larvae. Only a few LdE74 RNAi larvae finally became deformed pupae, with shortened antennae and legs. Therefore, LdE74 is required for larval-pupal metamorphosis and appendage growth in L. decemlineata.

SUMOylation is an important post-translational modification process that regulates different cellular functions in eukaryotes. SIZ/PIAS-type SAP and Miz1 (SIZ1) proteins exhibit SUMO E3 ligase activity, which modulates SUMOylation. However, SIZ1 in tomato has been rarely investigated. In this study, a tomato SIZ1 gene (SlSIZ1) was isolated and its molecular characteristics and role in tolerance to drought stress are described. SlSIZ1 was up-regulated by cold, sodium chloride (NaCl), polyethylene glycol (PEG), hydrogen peroxide (H2 O2 ) and abscisic acid (ABA), and the corresponding proteins were localized in the nucleus. The expression of SlSIZ1 in Arabidopsis thaliana (Arabidopsis) siz1-2 mutants partially complemented the phenotypes of dwarf, cold sensitivity and ABA hypersensitivity. SlSIZ1 also exhibited the activity of SUMO E3 ligase to promote the accumulation of SUMO conjugates. Under drought stress, the ectopic expression of SlSIZ1 in transgenic tobacco lines enhanced seed germination and reduced the accumulation of reactive oxygen species. SlSIZ1 overexpression conferred the plants with improved growth, high free proline content, minimal malondialdehyde accumulation and increased accumulation of SUMO conjugates. SlSIZ1 is a functional homolog of Arabidopsis SIZ1 with SUMO E3 ligase activity. Therefore, overexpression of SlSIZ1 enhanced the tolerance of transgenic tobacco to drought stress.

BACKGROUND Juvenile hormone (JH) plays a critical role in the regulation of metamorphosis in Leptinotarsa decemlineata, a notorious defoliator of potato. JH acid methyltransferase (JHAMT) is involved in one of the final steps of JH biosynthesis. RESULTS A putative JHAMT cDNA (LdJHAMT) was cloned. Two double-stranded RNAs (dsRNAs) (dsJHAMT1 and dsJHAMT2) against LdJHAMT were constructed and bacterially expressed. Experiments were conducted to investigate the effectiveness of RNAi in both second- and fourth-instar larvae. Dietary introduction of dsJHAMT1 and dsJHAMT2 successfully knocked down the target gene, lowered JH titre in the haemolymph and reduced the transcript of Kruppel homologue 1 gene. Ingestion of dsJHAMT caused larval death and weight loss, shortened larval developmental period and impaired pupation. Moreover, the dsJHAMT-fed pupae exhibited lower adult emergence rates. The resulting adults weighed an average of 50 mg less than the control group, and the females did not deposit eggs. Application of pyriproxyfen to the dsJHAMT-fed insects rescued all the negative effects. CONCLUSIONS LdJHAMT expresses functional JHAMT enzyme. The RNAi targeting LdJHAMT could be used for control of L. decemlineata. © 2015 Society of Chemical Industry

CBF3, a known cold-inducible gene that encodes a transcription factor, was isolated from Arabidopsis thaliana and introduced into the potato (Solanum tuberosum cv. 'luyin NO.1') under the control of the CaMV35S promoter or the rd29A promoter. Our results revealed that temperature of 40 °C or higher can significantly induce AtCBF3 expression. After heat stress, the net photosynthetic rate (Pn ), the maximal photochemical efficiency of photosystem II (PSII) (Fv /Fm ) and the accumulation of the D1 protein were higher in the transgenic lines than in the wild-type (WT) line. Moreover, compared with the WT line, O2 (●-) and H2 O2 accumulation in the transgenic lines were reduced. A Q-PCR assay of a subset of the genes involved in photosynthesis and antioxidant defence further verified the above results. Interestingly, under heat stress conditions, the accumulation of heat-shock protein 70 (HSP70) increased in the WT line but decreased in the transgenic lines. These results suggest that potato plants ectopically expressing AtCBF3 exhibited enhanced tolerance to high temperature, which is associated with improved photosynthesis and antioxidant defence via induction of the expression of many stress-inducible genes. However, this mechanism may not depend upon the regulatory pathways in which HSP70 is involved.

The roles of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ1) were investigated using wild-type (WT) and sense transgenic tomatoes. The LeCDJ1 expression was upregulated by 38 °C, 42 °C, 45 °C, NaCl, PEG, methyl viologen (MV) and hydrogen peroxide (H2O2), but not by 30 °C and 35 °C. Meanwhile, LeCDJ1 was involved in the response of plants to abscisic acid (ABA). Under heat stress, the sense plants showed better growth, higher chlorophyll content, lower malondialdehyde (MDA) accumulation and relative electrical conductivity (REC), and also less PSII photoinhibition than WT. Interestingly, the sense plants treated with streptomycin (SM), an inhibitor of organellar translation, still showed higher maximum photochemistry efficiency of PSII (Fv/Fm) and D1 protein levels than the SM-untreated WT, suggesting that the protective effect of LeCDJ1 on PSII was, at least partially, independent of D1 protein synthesis. Furthermore, the relatively lower superoxide radical (O2(•-)) and H2O2 levels in the sense plants were considered to be due to the higher ascorbate peroxidase (APX) and superoxide dismutase (SOD) activity, which seemed unlikely dependent on their transcription level. These results indicated that LeCDJ1 overexpression facilitated heat tolerance in transgenic tomatoes.

Zeaxanthin (Z) has a role in the dissipation of excess excitation energy by participating in non-photochemical quenching (NPQ) and is essential in protecting the chloroplast from photooxidative damage. To investigate the physiological effects and functional mechanism of constitutive accumulation of Z in the tomato at salt stress-induced photoinhibition and photooxidation, antisense-mediated suppression of zeaxanthin epoxidase transgenic plants and the wild-type (WT) tomato were used. The ratio of Z/(V + A + Z) and (Z + 0.5A)/(V + A + Z) in antisense transgenic plants were maintained at a higher level than in WT plants under salt stress, but the value of NPQ in WT and transgenic plants was not significantly different under salt stress. However, the maximal photochemical efficiency of PSII (Fv/Fm) and the net photosynthetic rate (Pn) in transgenic plants decreased more slowly under salt stress. Furthermore, transgenic plants showed lower level of hydrogen peroxide (H(2)O(2)), superoxide anion radical (O(2)(•-)) and ion leakage, lower malondialdehyde content. Compared with WT, the content of D1 protein decreased slightly in transgenic plants under salt stress. Our results suggested that the constitutive accumulation of Z in transgenic tomatoes can alleviate salt stress-induced photoinhibition because of the antioxidant role of Z in the scavenging quenching of singlet oxygen and/or free radicals in the lipid phase of the membrane.

The purpose of this study was to explore how the mitochondrial AOX (alternative oxidase) pathway alleviates photoinhibition in Rumex K-1 leaves. Inhibition of the AOX pathway decreased the initial activity of NADP-malate dehydrogenase (EC 1.1.1.82, NADP-MDH) and the pool size of photosynthetic end electron acceptors, resulting in an over-reduction of the photosystem I (PSI) acceptor side. The over-reduction of the PSI acceptor side further inhibited electron transport from the photosystem II (PSII) reaction centers to the PSII acceptor side as indicated by an increase in V(J) (the relative variable fluorescence at J-step), causing an imbalance between photosynthetic light absorption and energy utilization per active reaction center (RC) under high light, which led to the over-excitation of the PSII reaction centers. The over-reduction of the PSI acceptor side and the over-excitation of the PSII reaction centers enhanced the accumulation of reactive oxygen species (ROS), which inhibited the repair of the photodamaged PSII. However, the inhibition of the AOX pathway did not change the level of photoinhibition under high light in the presence of the chloroplast D1 protein synthesis inhibitor chloramphenicol, indicating that the inhibition of the AOX pathway did not accelerate the photodamage to PSII directly. All these results suggest that the AOX pathway plays an important role in the protection of plants against photoinhibition by minimizing the inhibition of the repair of the photodamaged PSII through preventing the over-production of ROS.

Tomato (Lycopersicon esculentum cv. 'Moneymaker') was transformed with a codA gene, from Arthrobacter globiformis, for choline oxidase that had been modified to allow targeting to both chloroplasts and the cytosol. Glycinebetaine (GB) accumulated in seeds of transformed plants up to 1 µmol g(-1) dry weight (DW), while no detectable GB was found in wild-type (WT) seeds. The codA-transgenic seeds germinated faster and at higher frequency than WT seeds with high temperature treatment. After heat stress, levels of expression of a mitochondrial small heat-shock protein (MT-sHSP), heat-shock protein 70 (HSP70) and heat-shock cognate 70 (HSC70) were higher in transgenic seeds than in WT seeds during heat stress, and the accumulation of HSP70 was more prominent in codA-transgenic seeds than in WT seeds. Addition of GB to the germination medium or imbibition of seeds in a solution of GB enhanced the tolerance of WT seeds to high temperatures. WT seeds treated with exogenous GB also expressed heat-shock genes at elevated levels and accumulated more HSP70 than controls. Our results suggest that GB, either applied exogenously or accumulated in vivo in codA-transgenic seeds, enhanced the expression of heat-shock genes in and improved the tolerance to high temperature of tomato seeds during germination.

An endoplasmic reticulum-localized tomato omega-3 fatty acid desaturase gene (LeFAD3) was isolated. The antisense tomato plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern blot analysis confirmed that the expression of LeFAD3 was inhibited in the tomato genome. Levels of 18:3 decreased and correspondingly levels of 18:2 increased in total lipids of leaves and roots. After heat stress, the fresh weight of the aerial parts of antisense transgenic plants was higher than that of the wild type (WT) plants. The membrane system ultrastructure of chloroplasts in leaf cells and all of the subcellular organelles in the root tips of transgenic plants remained more intact than those of WT. Relative electric conductivity increased less in transgenic plants than in WT. Under heat stress, the maximal photochemical efficiency of photosystem II (Fv/Fm) and the O(2) evolution rate decreased more in WT than in transgenic plants. These results suggested that the depletion of LeFAD3 increased the saturation of fatty acids and alleviated high temperature stress.

The purpose of this study was to explore the mechanisms by which Alternaria alternata damages tobacco (Nicotiana tabacum) leaves. Treatment with A. alternata metabolic products enhanced senescence in leaves of different ages, as indicated by the significant decrease in chlorophyll, soluble protein, photosynthetic O(2) evolution and catalase (CAT, EC 1.11.1.6) activity as well as an increase in H(2)O(2) content. The induction of senescence by A. alternata metabolic products increased as the age of the leaves increased. A. alternata metabolic products greatly influenced the behavior of photosystem II (PSII) in the leaves: oxygen evolving complex (OEC) activity and electron transport from primary quinone electron acceptor of PS II (Q(A)) to secondary quinone electron acceptor of PS II (Q(B)) were both significantly inhibited. This inhibition also became more pronounced in older leaves. In vitro experiments revealed that, without the influence of natural senescence, the A. alternata metabolic products directly inhibited the activity of a commercial CAT solution and inhibited photosynthetic O(2) evolution, which resulted in excess PSII excitation pressure and an overaccumulation of H(2)O(2) in leaf segments. These results suggest that the significant declines in photosynthesis and CAT activity induced by the metabolic products of A. alternata were important contributors to the overaccumulation of reactive oxygen species (ROS), which accelerated senescence in tobacco leaves. The fact that the enhancement of senescence was getting more pronounced with the age of tobacco leaves might be related to the fact that older leaves already had higher H(2)O(2) levels and less antioxidant activity as reflected in lower CAT activity.

In order to investigate the relationship between the lipid composition in thylakoid membrane and thermostability of photosynthetic apparatus, tobacco transformed with sweet pepper sense glycerol-3-phosphate acyltransferase (GPAT) gene were used to analyze the lipid composition in thylakoid membrane, the net photosynthetic rate and chlorophyll fluorescence parameters under high temperature stress. The results showed that the saturated extent of monogalactosyldiacylglycerol (MGDG), sulfoquinovosyldiacylglycerol, digalactosyldiacylglycerol and phosphatidylglycerol in thylakoid membrane of transgenic tobacco T(1) lines increased generally. Particularly, the saturated extent in MGDG increased obviously by 16.2% and 12.0% in T(1)-2 and T(1)-1, respectively. With stress temperature elevating, the maximum efficiency of photosystem II (PSII) photochemistry (Fv/Fm), actual photochemical efficiency of PSII in the light (Phi(PSII)) and net photosynthetic rate (Pn) of the two lines and wild type tobacco plants decreased gradually, but those parameters decreased much less in transgenic plants. Even though the recovery process appeared differently in the donor and acceptor side of PSII in transgenic tobacco compared with wild-type plants, the entire capability of PSII recovered faster in transgenic tobacco, which was shown in the parameters of PI, Fv/Fm and Phi(PSII), as a result, the recovery of Pn was accelerated. Conclusively, we proposed that the increase in saturated extent of thylakoid membrane lipids in transgenic plants enhanced the stability of photosynthetic apparatus under high temperature stress.

A tomato (Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene (LeZE) was isolated. The deduced amino acid sequence of LeZE showed high identities with zeaxanthin epoxidase in other plant species. Northern blot analysis showed that the mRNA accumulation of LeZE in the wild-type (WT) was not induced by light and temperature but regulated by the diurnal rhythm. The sense transgenic plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern and western blot analysis confirmed that sense LeZE was transferred into the tomato genome and overexpressed. The ratio of (A + Z)/(V + A + Z) and the values of non-photochemical quenching were lower in transgenic plants than in WT plants under high light and chilling stress with low irradiance. The O(2) evolution rate and the maximal photochemical efficiency of PSII (Fv/Fm) in transgenic plants decreased more quickly during both stresses and recovered slower than that in WT under optimal conditions. These results suggested that overexpression of LeZE impaired the function of the xanthophyll cycle and aggravated PSII photoinhibition in tomato under high light and chilling stress.

A chloroplast-localized tomato (Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase (LeGPAT) gene was isolated. The antisense tomato plants were got under the control of the caulifower mosaic virus 35S promoter (35S-CaMV). RNA gel blot analysis confirmed that the expression of LeGPAT was inhibited in the tomato genome. The depletion of LeGPAT caused a massive arrest in pollen development. It also increased the size of tapetal cells, delayed tapetum degeneration, reduced ER membrane biogenesis and altered oil body size. Results therefore suggested that LeGPAT played a crucial role in pollen development. There was, however, no effect on the ovule. The depletion of LeGPAT also increased the saturation of phosphatidylglycerol (PG) fatty acids in thylakoid membranes. Increase of PG-saturated fatty acids was helpful in alleviating photoinhibition of PSII in tomato plants under heat stress.

A chloroplast-localized tomato (Lycopersicon esculentum Mill.) ω-3 fatty acid desaturase gene (LeFAD7) was isolated and characterized with regard to its sequence, response to various temperatures, and function in antisense transgenic tomato plants. The deduced amino acid sequence had four histidine-rich regions, of which three regions were highly conserved throughout the whole ω-3 fatty acid desaturase gene family. Southern blotting analysis showed that LeFAD7 was encoded by a single copy gene and had two homologous genes in the tomato genome. Northern blot showed that LeFAD7 was expressed in all organs and was especially abundant in leaf tissue. Meanwhile, expression of LeFAD7 was induced by chilling stress (4 °C), but was inhibited by high temperature (45 °C), in leaves. Transgenic tomato plants were produced by integration of the antisense LeFAD7 DNA under the control of a CaMV35S promoter into the genome. Antisense transgenic plants with lower 18:3 content could maintain a higher maximal photochemical efficiency (Fv/Fm) and O2 evolution rate than wild-type plants. These results suggested that silence of the LeFAD7 gene alleviated high-temperature stress. There was also a correlation between the low content of 18:3 resulting from silence of the LeFAD7 gene and tolerance to high-temperature stress. (Managing editor: Li-Hui Zhao)