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Heat stress limits plant growth, development, and crop yield, but how plant cells precisely sense and transduce heat stress signals remains elusive. Here, we identified a conserved heat stress response mechanism to elucidate how heat stress signal is transmitted from the cytoplasm into the nucleus for epigenetic modifiers. We demonstrate that HISTONE DEACETYLASE 9 (HDA9) transduces heat signals from the cytoplasm to the nucleus to play a positive regulatory role in heat responses in Arabidopsis . Heat specifically induces HDA9 accumulation in the nucleus. Under heat stress, the phosphatase PP2AB′β directly interacts with and dephosphorylates HDA9 to protect HDA9 from 26S proteasome-mediated degradation, leading to the translocation of nonphosphorylated HDA9 to the nucleus. This heat-induced enrichment of HDA9 in the nucleus depends on the nucleoporin HOS1. In the nucleus, HDA9 binds and deacetylates the target genes related to signaling transduction and plant development to repress gene expression in a transcription factor YIN YANG 1–dependent and –independent manner, resulting in rebalance of plant development and heat response. Therefore, we uncover an HDA9-mediated positive regulatory module in the heat shock signal transduction pathway. More important, this cytoplasm-to-nucleus translocation of HDA9 in response to heat stress is conserved in wheat and rice, which confers the mechanism significant implication potential for crop breeding to cope with global climate warming.

Although much is known about plant responses to heat shock (HS), how plants sense high temperature and the primary HS signal transduction pathway leading to HS-regulated gene expression are still poorly understood. To identify primary transcription factors that mediate HS-regulated gene expression and their target genes, RNA sequencing was performed to detect genes whose expression is rapidly altered by HS in Arabidopsis (Arabidopsis thaliana). The results showed several genes were induced after only 5 min of HS treatment, suggesting that HS signaling occurs very rapidly. Analysis of the cis-elements in the promoters of genes upregulated by 10 min of HS treatment identified HEAT SHOCK FACTOR A1s (HSFA1s) and circadian clock proteins REVEILLE4 (RVE4) and RVE8 as essential transcription factors that independently mediate early HS-induced gene expression. Using hsfa1a/b/d/e and rve4/8 mutants, we identified subsets of HSFA1s- or RVE4/8-dependent early HS-induced genes and showed RVE4/8 regulate plant thermotolerance partially by regulating the expression of downstream transcription factors ETHYLENE RESPONSIVE FACTOR53 (ERF53) and ERF54, specifically around noon. These findings reveal a potential transcriptional regulatory hierarchy governing the first wave of HS-induced gene expression. They also provided important insight into the mechanism by which the circadian clock gates thermotolerance and prepares plants for exposure to high temperatures during the day.

In recent years, electrochromic materials have attracted much attention for application in many fields, such as smart windows, mirrors, optical displays, and camouflage materials. Due to the advantages of short switching time, easy color-tuning, and high optical contrast ratio, polymer materials have great value for this research. In this work, a series of nonconjugated poly(aryl amino ketone) (PAAK)-based materials with carbazol-9-yl triphenylamine (CzTPA) units were designed and synthesized. These polymers were first used to research electrochromism (EC) and electrofluorochromism (EFC). In spite of the fact that they are nonconjugated polymers, they still exhibited some exciting electrochromic and electrofluorochromic performances, such as good switching time (the fastest speed can reach 1.91 s for coloring and 0.66 s for bleaching), high optical contrast (PAAK-tBu-CzTPA's highest Δ%T is 87% at 730 nm) and rapid fluorescence response (1.42 s for quenching).

In this work, a room temperature atmospheric pressure direct-current plasma has been deployed for the one-step synthesis of gold nanoparticle/carboxyl group functionalized carbon nanotube (AuNP/CNT-COOH) nanohybrids in aqueous solution for the first time. Uniformly distributed AuNPs are formed on the surface of CNT-COOH, without the use of reducing agents or surfactants. The size of the AuNP can be tuned by changing the gold salt precursor concertation. UV-Vis, ζ-potential and X-ray photoelectron spectroscopy suggest that carboxyl surface functional groups on CNTs served as nucleation and growth sites for AuNPs and the multiple potential reaction pathways induced by the plasma-chemistry have been elucidated in detail. The nanohybrids exhibit significantly enhanced Raman scattering and photothermal conversion efficiency, properties that are essential for potential multi-modal cancer treatment applications

In this work, we designed and fabricated a multifunctional nanocomposite system that consists of chitosan, raspberry-like silver nanoparticles, and graphene oxide. The room temperature atmospheric pressure microplasma (RT-APM) process provides a rapid, facile, and environmentally-friendly method for introducing silver nanoparticles into the composite system. Our composite can achieve a pH controlled single and/or dual drug release. Under pH 7.4 for methyl blue loaded on chitosan, the drug release profile features a burst release during the first 10 h, followed by a more stabilized release of 70–80% after 40–50 h. For fluorescein sodium loaded on graphene oxide, the drug release only reached 45% towards the end of 240 h. When the composite acted as a dual drug release system, the interaction of fluorescein sodium and methyl blue slowed down the methyl blue release rate. Under pH 4, both single and dual drug systems showed a much higher release rate. In addition, our composite system demonstrated strong antibacterial abilities against E. coli and S. aureus, as well as an excellent photothermal conversion effect under irradiation of near infrared lasers. The photothermal conversion efficiency can be controlled by the laser power. These unique functionalities of our nanocomposite point to its potential application in multiple areas, such as multimodal therapeutics in healthcare, water treatment, and anti-microbials, among others.

Background As global warming becomes increasingly severe, it is urgent that we enhance the heat tolerance of crops. We previously reported that Arabidopsis thaliana PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE C9 (AtPLC9) promotes heat tolerance. Results In this study, we ectopically expressed AtPLC9 in rice to examine its potential to improve heat tolerance in this important crop. Whereas AtPLC9 did not improve rice tolerance to salt, drought or cold, transgenic rice did exhibit greater heat tolerance than the wild type. High-throughput RNA-seq revealed extensive and dynamic transcriptome reprofiling in transgenic plants after heat stress. Moreover, the expression of some transcription factors and calcium ion-related genes showed specific upregulation in transgenic rice after heat stress, which might contribute to the enhanced heat tolerance. Conclusions This study provides preliminary guidance for using AtPLC9 to improve heat tolerance in cereal crops and, more broadly, highlights that heterologous transformation can assist with molecular breeding.

In recent years, plasma-induced non-equilibrium electrochemistry (PiNE) has been increasingly used for the synthesis of nanomaterials. In this study, we investigated the effect of solution pH on the formation of AuNP/MWCNT nanocomposites synthesized by PiNE. It is found that resulting nanocomposite morphology can be manipulated by the solution pH with pH 2 giving the most uniformly distributed AuNP along the MWCNT surface during the nanocomposite formation. The detailed mechanisms of AuNP/MWCNT nanocomposites formation under different pH have been discussed. For selected AuNP/MWCNT, we further evaluated the photothermal conversion performance under a blue laser (wavelength 445 nm) and the material biocompatibility using HeLa cells. The promising photothermal capability and biocompatibility of the composite sample point to their potential future applications such as solar thermal conversion and healthcare technology.

In this paper, an experimental study was decided to investigate the dependence of tribological property on biphenyl group contents in the polymer chain. A series of biphenyl containing poly (ether-ether-ketone-ketone) copolymers were synthesized by nucleophilic substitution polycondensation, the contents of biphenyl units were controlled by the feed ratio of biphenol to hydroquinone. The sliding wear and friction tests were conducted using a pin-on-disc apparatus against a stainless steel counterpart without any lubricant under dry conditions. The results indicated the friction coefficient decreased with the growing biphenyl contents first and then increased after BP60, associated with the same change of polymer crystallinity, while the specific wear rate exhibited a negative correlation. It was demonstrated that this bridge between polymer structure, crystallinity, and tribological performance was independent of the test conditions. For one specific sample, raising applied load and sliding speed will aggravate the wear work, while the friction coefficient was only distinctly affected by the applied load, having no concern with the sliding speed. Probable mechanisms were discussed by observation of the worn surface using scanning electron microscopy. Within our efforts, a possible molecular design method was proposed to develop potential tribological resins via adjusting one specific group in the backbone. Keywords: Tribological polymers, Sliding friction, Sliding wear, Molecular design, Biphenyl group

ROOT MERISTEM GROWTH FACTOR (RGF) 1 is an important peptide hormone that regulates root growth. Upon binding to its receptor, RGFR1, RGF1 regulates the expression of two transcription factors, PLETHORA 1 and 2 (PLT1/2), to influence root meristem development. Here, we show that the ubiquitin-specific proteases UBP12 and UBP13 are positive regulators of root meristem development and that UBP13 interacts directly with RGF1 receptor (RGFR1) and its close homolog RGFR2. The ubp12,13 double-mutant root is completely insensitive to exogenous applied RGF1. Consistent with this result, RGF1-induced ubiquitination and turnover of RGFR1 protein were accelerated in ubp12,13-mutant plants but were delayed in transgenic plants overexpressing UBP13 Genetic analysis showed that PLT2 or RGFR1 overexpression partially rescued the short-root phenotype and the reduced cortical root meristem cell number in ubp12,13 plants. Together, our results demonstrate that UBP12/13 are regulators of the RGF1-RGFR1-PLT1/2 signaling pathway and that UBP12/13 can counteract RGF1-induced RGFR1 ubiquitination, stabilize RGFR1, and maintain root cell sensitivity to RGF1.

Cell-to-cell communication precisely controls the creation of new organs during reproductive growth. However, the sensor molecules that mediate developmental signals in monocot plants are poorly understood. Here, we report that DWARF AND RUNTISH SPIKELET1 (DRUS1) and DRUS2, two closely related receptor-like kinases (RLKs), redundantly control reproductive growth and development in rice (Oryza sativa). A drus1-1 drus2 double knockout mutant, but not either single mutant, showed extreme dwarfism and barren inflorescences that harbored sterile spikelets. The gibberellin pathway was not impaired in this mutant. A phenotypic comparison of mutants expressing different amounts of DRUS1 and 2 revealed that reproductive growth requires a threshold level of DRUS1/2 proteins. DRUS1 and 2 maintain cell viability by repressing protease-mediated cell degradation and likely by affecting sugar utilization or conversion. In the later stages of anther development, survival of the endothecium requires DRUS1/2, which may stimulate expression of the UDP-glucose pyrophosphorylase gene UGP2 and starch biosynthesis in pollen. Unlike their Arabidopsis thaliana ortholog FERONIA, DRUS1 and 2 mediate a fundamental signaling process that is essential for cell survival and represents a novel biological function for the CrRLK1L RLK subfamily.

The receptor-like kinase SIT1 acts as a sensor in rice (Oryza sativa) roots, relaying salt stress signals via elevated kinase activity to enhance salt sensitivity. Here, we demonstrate that Protein Phosphatase 2A (PP2A) regulatory subunit B'κ constrains SIT1 activity under salt stress. B'κ-PP2A deactivates SIT1 directly by dephosphorylating the kinase at Thr515/516, a salt-induced phosphorylation site in the activation loop that is essential for SIT1 activity. B'κ overexpression suppresses the salt sensitivity of rice plants expressing high levels of SIT1, thereby contributing to salt tolerance. B'κ functions in a SIT1 kinase-dependent manner. During early salt stress, activated SIT1 phosphorylates B'κ; this not only enhances its binding with SIT1, it also promotes B'κ protein accumulation via Ser502 phosphorylation. Consequently, by blocking SIT1 phosphorylation, B'κ inhibits and fine-tunes SIT1 activity to balance plant growth and stress adaptation.

This is the first study on the deployment of direct current atmospheric pressure microplasma technique for the single step synthesis of gold nanoparticle/graphene oxide (AuNP/GO) nanocomposites. The nanocomposites were characterized using ultraviolet–visible spectroscopy (UV–vis), x-ray diffraction and x-ray photoelectron spectroscopy and their formation mechanisms have been discussed in detail. Our AuNP/GO nanocomposites are highly biocompatible and have demonstrated surface enhanced Raman scattering (SERS) properties as compared to pure AuNPs and pure GO. Their potential as SERS substrate has been further demonstrated using probe molecules (methylene blue) at different concentrations.

To understand the early heat shock (HS)-regulated cellular responses that influence the tolerance of rice plant to high environmental temperatures, two-dimensional difference gel electrophoresis (2D-DIGE) is performed to explore the early HS-regulated proteome. Multiple proteins that show abundance changes after 1 and 5 min of HS treatment are identified. Of the early HS-regulated proteins identified, the abundance of a ubiquitin-specific protease, OsUBP21, and its Arabidopsis homolog, AtUBP13, is found to be upregulated by 5 min of HS treatment. Further, knocking the expression of OsUBP21 or AtUBP13 down or out increases the tolerance of rice and Arabidopsis plants to HS stress, suggesting that the function of these ubiquitin-specific proteases in regulating plant HS responses is conserved between monocots and dicots. 2D-DIGE showed a group of proteins are differentially regulated in wild-type and ubp21 mutant after 30 min of HS treatment. Among these proteins, 11 are found to interact directly with OsUBP21; thus, they may be targets of OsUBP21. Future analyses of the roles of these OsUBP21-interacting proteins in plant HS responses will help reveal the protein ubiquitination/deubiquitination-regulated cellular responses induced by HS in rice.

In plants, flowering time is controlled by environmental signals such as day-length and temperature, which regulate the floral pathway integrators, including FLOWERING LOCUS T (FT), by genetic and epigenetic mechanisms. Here, we identify an H3K27me3 demethylase, JUMONJI 13 (JMJ13), which regulates flowering time in Arabidopsis. Structural characterization of the JMJ13 catalytic domain in complex with its substrate peptide reveals that H3K27me3 is specifically recognized through hydrogen bonding and hydrophobic interactions. Under short-day conditions, the jmj13 mutant flowers early and has increased FT expression at high temperatures, but not at low temperatures. In contrast, jmj13 flowers early in long-day conditions regardless of temperature. Long-day condition and higher temperature induce the expression of JMJ13 and increase accumulation of JMJ13. Together, our data suggest that the H3K27me3 demethylase JMJ13 acts as a temperature- and photoperiod-dependent flowering repressor., Jumonji domain-containing histone demethylases regulate flowering in plants. Here Zheng et al. show that Arabidopsis JMJ13 is an H3K27me3 demethylase that recognizes H3K27me3 via hydrogen bonding and hydrophobic interactions and affects both photoperiod and temperature-dependent flowering responses.

A novel triphenylamine-containing polyarylsulfone (PAS-TPA) was synthesized from commercially available aniline and 1,1′-sulfonylbis(4-fluorobenzene) via two-step N–C coupling reaction under nucleophilic substitution polycondensation condition. Subsequently, a new series of polysulfones with different TPA contents (PSU-TPAs) was developed by the same method. Both PAS-TPA and PSU-TPAs not only exhibited good thermal stability and solubility but also exhibited intense ultraviolet absorption and fluorescence and high fluorescence quantum yield (approximately 16–42%). Their ideal highest occupied molecular orbital level (approximately −5.5 to −5.6) indicated the potential application of these polymers in organic photoelectronic fields.

To understand the early signaling steps that regulate cold responses in rice, two-dimensional difference gel electrophoresis (2-D DIGE)1 was used to study early cold-regulated proteins in rice seedlings. Using mass spectrometry, 32 spots, which represent 26 unique proteins that showed an altered expression level within 5 min of cold treatment were identified. Among these proteins, Western blot analyses confirmed that the cellular phospholipase D α1 (OsPLDα1) protein level was increased as early as 1 min after cold treatment. Genetic studies showed that reducing the expression of OsPLDα1 makes rice plants more sensitive to chilling stress as well as cold acclimation increased freezing tolerance. Correspondingly, cold-regulated proteomic changes and the expression of the cold-responsive C repeat/dehydration-responsive element binding 1 (OsDREB1) family of transcription factors were inhibited in the pldα1 mutant. We also found that the expression of OsPLDα1 is directly regulated by OsDREB1A. This transcriptional regulation of OsPLDα1 could provide positive feedback regulation of the cold signal transduction pathway in rice. OsPLDα1 hydrolyzes phosphatidylcholine to produce the signal molecule phosphatidic acid (PA). By lipid-overlay assay, we demonstrated that the rice cold signaling proteins, MAP kinase 6 (OsMPK6) and OsSIZ1, bind directly to PA. Taken together, our results suggest that OsPLDα1 plays a key role in transducing cold signaling in rice by producing PA and regulating OsDREB1s' expression by OsMPK6, OsSIZ1, and possibly other PA-binding proteins.

Perfluorocarbon groups or related compounds are usually used to modify polymer materials because of their low surface energy properties. In this paper, a novel perfluorocarbon-containing compound was synthesized and used as a capping agent for the preparation of perfluorocarbon chain terminated poly(ether sulfone). The regulation of the fluorine content was achieved by controlling the molecular weight. FTIR and 1H NMR spectra were used to confirm the existence of perfluorocarbon terminated groups in the polymer chains. In addition, the ratios of F/C were acquired by integral area calculation from the 1H NMR spectrum. XPS characterization showed that the surface migration phenomenon of fluorine became apparent along with the increase of the F/C ratio. Further testing found that the introduction of perfluorinated end groups into the PES chains had little influence on the polymers' mechanical and thermal performance, by contrast, their surface energy decreased efficiently from approximately 40 mJ m−2 to 16 mJ m−2 with the F/C ratio on the surface increased from 0 to 0.556, which could develop PES as a low surface energy material for industrial applications.

Room temperature atmospheric pressure microplasma (APM) was deployed for the first time for the in situ synthesis of anti-bacterial silver nanoparticle / chitosan (AgNP/CS) nanocomposites. The plasma induced liquid chemistry plays a role in the in situ formation of AgNP, the size distribution of which depends on the silver salt precursor concentration. The microplasma process has also simultaneously tailored the physical properties of the composites, rendering more crosslinked chitosan polymer network with shorter molecular chains. The formation of AgNP within the in situ modified chitosan has led to nanocomposites with overall improved mechanical properties and better stability in simulated body fluid. Our plasma synthesized AgNP/CS nanocomposites also demonstrate effective antibacterial properties against E. Coli and S. Aureus bacterial strains, showing their promise in potential antimicrobial applications.

A novel homopolyarylsulfone with 4-(carbazol-9-yl) triphenylamine moieties was synthesized successfully through N–C coupling reactions under similar conditions to the synthesis of polyarylsulfones (PSU). By using this simple method and adjusting the feed ratio between 9-(4-aminobenzene) carbazole (NH2–Ar–Cz) and bisphenol A, a series of high-performance copolymers with high molecular weight, good film-reforming ability, great thermal stability, and excellent photoelectric characteristics were also prepared, which have good application potential in the field of organic optoelectronics.

High-molecular-weight poly(arylene ether kekone) (PAEK) containing 3-(trifluoromethyl) phenyl pendants and biphenyl units with a mole ratio of 3:7 (FP30-PEDEKK) has been synthesized by nucleophilic substitution copolymerization from 3-(trifluromethyl) phenylhydroquinone, 4,4′-biphenol, and 1,4-bis(p-flurobenzoyl) benzene. FP30-PEDEKK shows good mechanical strength and high thermal stability. The friction and wear behaviors of the copolymer were investigated on pin-on-disk tribological test method using UMT-2. Compared to poly(ether ether ketone) (PEEK), FP30-PEDEKK has better friction reduction property and more stable friction behavior, although it shows lower wear resistance. These results could be supported by the scanning electron microscope investigations which showed that FP30-PEDEKK did have smoother worn surface than PEEK under given conditions. From microscopic images and the evolution of the friction coefficient and specific wear rate, the wear mechanisms of FP30-PEDEKK were suggested. This work significantly highlights the potential of molecular design as a route toward the tribological development of PAEK.

Calcium ions (Ca(2+)) play a crucial role in many key physiological processes; thus, the maintenance of Ca(2+) homeostasis is of primary importance. Na(+)/Ca(2+) exchangers (NCXs) play an important role in Ca(2+) homeostasis in animal excitable cells. Bioinformatic analysis of the Arabidopsis genome suggested the existence of a putative NCX gene, Arabidopsis NCX-like (AtNCL), encoding a protein with an NCX-like structure and different from Ca(2+)/H(+) exchangers and Na(+)/H(+) exchangers previously identified in plant. AtNCL was identified to localize in the Arabidopsis cell membrane fraction, have the ability of binding Ca(2+), and possess NCX-like activity in a heterologous expression system of cultured mammalian CHO-K1 cells. AtNCL is broadly expressed in Arabidopsis, and abiotic stresses stimulated its transcript expression. Loss-of-function atncl mutants were less sensitive to salt stress than wild-type or AtNCL transgenic overexpression lines. In addition, the total calcium content in whole atncl mutant seedlings was higher than that in wild type by atomic absorption spectroscopy. The level of free Ca(2+) in the cytosol and Ca(2+) flux at the root tips of atncl mutant plants, as detected using transgenic aequorin and a scanning ion-selective electrode, required a longer recovery time following NaCl stress compared with that in wild type. All of these data suggest that AtNCL encodes a Na(+)/Ca(2+) exchanger-like protein that participates in the maintenance of Ca(2+) homeostasis in Arabidopsis. AtNCL may represent a new type of Ca(2+) transporter in higher plants.

Plants respond and adapt to temperature changes by many ways. In this article, we provide a supplement to previous work about AtPLC3. The subcellular localization showed that AtPLC3 was located in the plasma membrane and nucleus which differed from AtPLC9 localization. Furthermore, we measured the contents of IP3 before and after HS in AtPLC3 mutant, complemented and overexpressing lines. The results showed that the increase in IP3 after HS was partially dependent on AtPLC3 activity. To sum up, the similar expression patterns and phenotypes suggested that AtPLC3 and AtPLC9 may regulate the thermotolerance of Arabidopsis by the same mechanisms.

An increased concentration of cytosolic calcium ions (Ca²⁺) is an early response by plant cells to heat shock. However, the molecular mechanism underlying the heat-induced initial Ca²⁺ response in plants is unclear. In this study, we identified and characterized a heat-activated Ca²⁺-permeable channel in the plasma membrane of Arabidopsis thaliana root protoplasts using reverse genetic analysis and the whole-cell patch-clamp technique. The results indicated that A. thaliana cyclic nucleotide-gated ion channel 6 (CNGC6) mediates heat-induced Ca²⁺ influx and facilitates expression of heat shock protein (HSP) genes and the acquisition of thermotolerance. GUS and GFP reporter assays showed that CNGC6 expression is ubiquitous in A. thaliana, and the protein is localized to the plasma membrane of cells. Furthermore, it was found that the level of cytosolic cAMP was increased by a mild heat shock, that CNGC6 was activated by cytosolic cAMP, and that exogenous cAMP promoted the expression of HSP genes. The results reveal the role of cAMP in transduction of heat shock signals in plants. The correlation of an increased level of cytosolic cAMP in a heat-shocked plant with activation of the Ca²⁺ channels and downstream expression of HSP genes sheds some light on how plants transduce a heat stimulus into a signal cascade that leads to a heat shock response.

The palea and lemma are unique organs in grass plants that form a protective barrier around the floral organs and developing kernel. The interlocking of the palea and lemma is critical for maintaining fertility and seed yield in rice; however, the molecules that control the interlocking structure remain largely unknown. Here, we showed that when OsCR4 mRNA expression was knocked down in rice by RNA interference, the palea and lemma separated at later spikelet stages and gradually turned brown after heading, resulting in the severe interruption of pistil pollination and damage to the development of embryo and endosperm, with defects in aleurone. The irregular architecture of the palea and lemma was caused by tumour-like cell growth in the outer epidermis and wart-like cell masses in the inner epidermis. These abnormal cells showed discontinuous cuticles and uneven cell walls, leading to organ self-fusion that distorted the interlocking structures. Additionally, the faster leakage of chlorophyll, reduced silica content and elevated accumulation of anthocyanin in the palea and lemma indicated a lesion in the protective barrier, which also impaired seed quality. OsCR4 is an active receptor-like kinase associated with the membrane fraction. An analysis of promoter::GUS reporter plants showed that OsCR4 is specifically expressed in the epidermal cells of paleas and lemmas. Together, these results suggest that OsCR4 plays an essential role in maintaining the interlocking of the palea and lemma by promoting epidermal cell differentiation.

Intracellular calcium (Ca(2+)) increases rapidly after heat shock (HS) in the Ca(2+)/calmodulin (Ca(2+)/CaM) HS signal transduction pathway: a hypothesis proposed based on our previous findings. However, evidence for the increase in Ca(2+) after HS was obtained only through physiological and pharmacological experiments; thus, direct molecular genetic evidence is needed. The role of phosphoinositide-specific phospholipase C (PI-PLC) is poorly understood in the plant response to HS. In this work, atplc9 mutant plants displayed a serious thermosensitive phenotype compared with wild-type (WT) plants after HS. Complementation of atplc9 with AtPLC9 rescued both the basal and acquired thermotolerance phenotype of the WT plants. In addition, thermotolerance was even improved in overexpressed lines. The GUS staining of AtPLC9 promoter:GUS transgenic seedlings showed that AtPLC9 expression was ubiquitous. The fluorescence distribution of the fusion protein AtPLC9 promoter:AtPLC9:GFP revealed that the subcellular localization of AtPLC9 was restricted to the plasma membrane. The results of a PLC activity assay showed a reduction in the accumulation of inositol-1,4,5-trisphosphate (IP(3)) in atplc9 during HS and improved IP(3) generation in the overexpressed lines. Furthermore, the heat-induced increase in intracellular Ca(2+) was decreased in atplc9. Accumulation of the small HS proteins HSP18.2 and HSP25.3 was downregulated in atplc9 and upregulated in the overexpressed lines after HS. Together, these results provide molecular genetic evidence showing that AtPLC9 plays a role in thermotolerance in Arabidopsis.

A novel heat shock (HS) signal transduction pathway in plants for the participation of Ca2+–calmodulin (CaM) in HS signal transduction was identified. HS induces a rapid increase in intracellular free calcium ion levels ([Ca2+]i), and the involvement of phospholipase C-inositol 1,4,5-trisphosphate is one of the factors leading to elevation in [Ca2+]i induced by HS. HS also increases the expression of the CaM gene and the accumulation of the CaM protein. The CaM isoform, AtCaM3, in Arabidopsis is a key member in the HS signal transduction pathway. AtCaM3 regulates the activity of CaM-binding protein kinase (AtCBK3) or protein phosphatase (AtPP7), promoting the activation of the HS transcription factor, AtHSFA1a, by phosphorylation/dephosphorylation and the expression of heat shock protein genes, then improving heat tolerance in plants.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) comprise the largest subfamily of transmembrane receptor-like kinases in plants, and they regulate a wide variety of developmental and defense-related processes. In this study, RNA interference (RNAi) strategy was used to specifically knockdown 59 individual rice genes encoding putative LRR-RLKs, and a novel rice blast resistance-related gene (designated as OsBRR1) was identified by screening T(0) RNAi population using a weakly virulent isolate of Magnaporthe oryzae, Ken 54-04. Wild-type plants (Oryza sativa L. cv. 'Nipponbare') showed intermediate resistance to Ken 54-04, while OsBRR1 suppression plants were susceptible to Ken 54-04. Furthermore, OsBRR1-overexpressing plants exhibited enhanced resistance to some virulent isolates (97-27-2, 99-31-1 and zhong 10-8-14). OsBRR1 expression was low in leaves and undetectable in roots under normal growth conditions, while its transcript was significantly induced in leaves infected with the blast fungus (Ken 54-04) and was moderately affected by ABA, JA and SA treatment. Overexpression or RNAi suppression of OsBRR1 did not cause visible developmental changes in rice plants. These results indicate that OsBRR1 is involved in rice resistance responses to blast fungus and mediates resistance to rice blast.

Heat shock (HS) is a common form of stress suffered by plants. It has been proposed that calmodulin (CaM) is involved in HS signal transduction, but direct evidence has been lacking. To investigate the potential regulatory function of CaM in the HS signal transduction pathway, T-DNA knockout mutants for AtCaM2, AtCaM3, and AtCaM4 were obtained and their thermotolerance tested. Of the three knockout mutant plants, there were no differences compared with wild-type plants under normal conditions. However, the AtCaM3 knockout mutant showed a clear reduction in thermotolerance after heat treatment at 45°C for 50 min. Overexpression of AtCaM3 in either the AtCaM3 knockout or wild-type background significantly rescued or increased the thermotolerance, respectively. Results from electrophoretic mobility-shift assays, real-time quantitative reverse transcription-polymerase chain reaction, and western-blot analyses revealed that, after HS, the DNA-binding activity of HS transcription factors, mRNA transcription of HS protein genes, and accumulation of HS protein were down-regulated in the AtCaM3 knockout mutant and up-regulated in the AtCaM3-overexpressing transgenic lines. Taken together, these results suggest that endogenous AtCaM3 is a key component in the Ca2+-CaM HS signal transduction pathway.

AtDjA2 and atDjA3 are members of J-domain molecular chaperone family of Arabidopsis thaliana. Analysis of the spatial expression of promoter:GUS fusions showed that both genes are expressed in young roots and leaves of seedlings, as well as in flowers and young siliques. The results from quantitative real-time PCR analyses indicate that the expression of both genes increased rapidly after a 37 °C heat shock. Similar results were seen when the promoter:GUS fusion plants were exposed to heat shock conditions. The heat shock-induced increase in atDjA3 gene expression was more rapid and to a greater extent than seen with the atDjA2 gene. Both T-DNA insertional KO lines and ectopic 35S-driven overexpressing lines of both genes were employed in an attempt to specific roles for the two genes. The individual T-DNA insertional mutants had no obvious physical phenotype, however, seedlings of double-KO lines had impaired thermotolerance. Conversely, ectopic overexpression of both genes resulted in plants with increased thermotolerance. These results suggest a conserved function for both genes, and a dose-dependent effect on seedling thermotolerance.

Lipid transfer proteins in plants are believed to be involved in many processes of cell physiology and development. In this work, a full-length cDNA encoding a novel lipid transfer protein, designated BcLTP was isolated from Brassica chinensis. At least two copies of BcLTP are present in whole genome of B. chinensis, and its transcripts preferably accumulate in second-year organs, implying its role in reproductive growth stage. The 118 amino acid sequence deduced from a 354 bp open reading frame (ORF) shares common features with other members of plants LTPs family. A putative signal peptide at the N terminus was tested for secretion function by the yeast signal sequence trap (YSST) system, and further confirmed by vesicular and extracellular localization of YFP fusion protein. A highly conserved CaM binding site at C terminus was found and the binding properties with two representative CaM isoforms, one is convergent AtCaM2, one is divergent SCaM5, were determined by gel overlay. We found that convergent AtCaM2 prefer high concentration of Ca(2+) for binding BcLTP, while SCaM5 does not depend on Ca(2+ )concentration too much for binding BcLTP. The lipid binding feature of BcLTP was demonstrated using florescence-marked 1-pyrenedodecanoic acid, which can be enhanced by AtCaM2 in Ca(2+ )dependent manner and by SCaM5 in either presence or absence of Ca(2+). The collected data suggest that BcLTP may secrete and combine extracellular CaM isoforms, which in turn, facilitate lipid binding of BcLTP via Ca(2+) mediated signaling.

To understand the early signaling steps in the response of plant cells to increased environmental temperature, 2-D difference gel electrophoresis was used to study the proteins in microsomes of Arabidopsis seedlings that are regulated early during heat stress. Using mass spectrometry, 19 microsomal proteins that showed an altered expression level within 5 min after heat treatment were identified. Among these proteins, annexin 1 (AtANN1) was one of those up-regulated rapidly after heat-shock treatment. Functional studies show loss-of-function mutants for AtANN1 and its close homolog AtANN2 were more sensitive to heat-shock treatment, whereas plants overexpressing AtANN1 showed more resistance to this treatment. Correspondingly, the heat-induced expression of heat-shock proteins and heat-shock factors is inhibited in ann1/ann2 double mutant, and the heat-activated increase in cytoplasmic calcium concentration ([Ca(2+)]cyt) is greatly impaired in the ann1 mutant and almost undetectable in ann1/ann2 double mutant. Taken together these results suggest that AtANN1 is important in regulating the heat-induced increase in [Ca(2+)]cyt and in the response of Arabidopsis seedlings to heat stress.

The involvement of calcium and different calmodulin isoforms (Ca(2+)-CaM) in heat shock (HS) signal transduction in Arabidopsis (Arabidopsis thaliana) was investigated. Using transgenic Arabidopsis plants which have the AtHsp18.2 promoter/GUS fusion gene, it was found that the level of beta-glucuronidase (GUS) activity was up-regulated by the addition of CaCl2 and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl3 and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), chlorpromazine (CPZ) and trifluoperazine (TFP). CaCl2 not only increased the GUS activity after HS, but also up-regulated the GUS activity under non-HS conditions. These results provide additional support for the involvement of the Ca(2+)-CaM signalling system in HSP gene expression. The expression of nine CaM genes (AtCaM1-9) from Arabidopsis was differentially regulated by HS at 37 degrees C. The expression of AtCaM3 and AtCaM7 genes increased during HS. The temporal expression of the AtCaM3, AtCaM7 and hsp18.2 genes demonstrated that up-regulation of AtCaM3 expression occurred earlier than that of AtCaM7 or hsp18.2.

Calmodulin, a highly conserved protein family that has long been well known as an intracellular calcium sensor, was identified in the culture medium and cell walls of Arabidopsis thaliana suspension-cultured cells by immunoblotting assay. A promotion effect by applying exogenous purified calmodulin and an inhibition effect by the addition of anti-calmodulin anti-serum or calmodulin antagonist to the medium on proliferation of suspension cells were found by monitoring incorporation of [methyl-3H]thymidine into nuclear DNA. Radioligand binding analysis with 35S-labeled calmodulin indicated the presence of specific, reversible, and saturable calmodulin binding sites on the surface of both A. thaliana suspension-cultured cells and its protoplasts; among them at least one is on the surface of Arabidopsis protoplasts, with the Kd approximately 9.2 nM, and two are on the out-surface of Arabidopsis suspension-cultured cells, with Kd values of approximately 47.5 and 830 nM. Chemical crosslinking of 35S-labeled calmodulin to protoplasts revealed 117- and 41-kDa plasma membrane proteins specifically bound to calmodulin, whereas cross-linking with intact suspension-cultured cells verified more calmodulin binding proteins which might be cell wall-associated in addition to membrane-localized. Taking together, our data provide first evidence for the presence of apoplastic calmodulin receptor-like binding proteins on the cell surface of Arabidopsis suspension-cultured cells, which strongly supports our previous idea that apoplastic calmodulin functions as a peptide signal involved in regulation of cell growth and development.

Confocal laser scanning microscopy (CLSM) and whole-cell patch-clamp were used to investigate the role of Ca2+ influx in maintaining the cytosolic Ca2+ concentration ([Ca2+]c) and the features of the Ca2+ influx pathway in germinating pollen grains of Lilium davidii D. [Ca2+]c decreased when Ca2+ influx was inhibited by EGTA or Ca2+ channel blockers. A hyperpolarization-activated Ca2+-permeable channel, which can be suppressed by trivalent cations, verapamil, nifedipine or diltiazem, was identified on the plasma membrane of pollen protoplasts with whole-cell patch-clamp recording. Calmodulin (CaM) antiserum and W7-agarose, both of which are cell-impermeable CaM antagonists, lead to a [Ca2+]c decrease, while exogenous purified CaM triggers a transient increase of [Ca2+]c and also remarkably activated the hyperpolarization-activated Ca2+ conductance on plasma membrane of pollen protoplasts in a dose-dependent manner. Both the increase of [Ca2+]c and the activation of Ca2+ conductance which were induced by exogenous CaM were inhibited by EGTA or Ca2+ channel blockers. This primary evidence showed the presence of a voltage-dependent Ca2+-permeable channel, whose activity may be regulated by extracellular CaM, in pollen cells.

Cytoplasmic free calcium concentration ([ Ca2+ ]c) in pollen cells of Lilium daviddi is measured with confocal laser scanning microscopy to investigate the effect of heterotrimeric G protein (G protein) on [Ca 2+ ]c and the possible signal transduction pathway of G protein triggering cellular calcium signal. After application, cholera toxin (CTX), an agonist of G protein, triggers a transient increase of [Ca ]c in pollen cells, and evokes a spatial-temporal characteristic calcium dynamics; while pertussis toxin (PTX), a G pro-tein antagonist, leads to the decrease of [Ca2+ ]c. Both L-type Ca2+ channel blocker verapamil and inhibitor of IP3 receptor heparin inhibit CTX-induced [Ca 2+ ]c increase. The results show that G protein may play a role in the modulation of [Ca2+ ]c through enhancing the extracellular Ca 2+ influx and releasing of Ca from intracellular stores.

The short-term effects of rare earth elements on pollen germination and tube growth were tested. Concentrations of 2.5∼20 µm lanthanum(La3+) or cerium (Ce3+)increased pollen germination and pollen tube growth, whereas concentrations higher than 40 µm La3+ and Ce3+ inhibited this process. The most effective concentration of La3+ needed for promotion shifted from 10 to 40 µm, depending on the Ca2+ concentration in the medium. Calmodulin (CaM) antagonist W7-agarose and anti-CaM antibody depressed La3+-promoted pollen germination and tube growth in a dose-dependent manner. La3+–CaM complexes (La3+–CaM) increased pollen germination and tube growth more than CaM or La3+ alone. Pertussis toxin (PTX) inhibited La3+-promoted pollen germination and tube growth. Cholera toxin (CTX) partially recovered the inhibition of the above La3+-promoted process by the anti-CaM antibody. Concentrations of 10−7∼10−9 m La3+–CaM increased GTPase activity inside plasma membrane vesicles of the pollen tube, but apo-CaM or La3+ alone had no positive effects. The results suggest that apoplastic CaM may be involved in the promotion effects of lower concentrations of La3+ on pollen germination and tube growth, and the heterotrimeric G-protein on the plasma membrane may transduce La3+-activated CaM signalling. The present studies provide an apoplastic mechanism for short-term effects of rare earth elements at lower concentrations in the pollen system.

The DNA-binding activity of heat shock transcription factor (HSF) was induced by heat shock (HS) of a whole cell extract. Addition of antiserum, specific to CaM, to a whole cell extract reduced bind of the HSF to the heat shock element (HSE) with maize, and the re-addition of CaM to the sample restored the activity of the HSF for binding to HSE. In addition, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract at non-HS temperature with maize. Similar results were obtained with wheat and tomato. Our observations provide the first example of the involvement of CaM in regulation of the DNA-binding activity of the HSF.

The heat stress response is an important adaptation, enabling plants to survive challenging environmental conditions. Our previous work demonstrated that Arabidopsis thaliana Phosphoinositide-Specific Phospholipase C Isoform 9 (AtPLC9) plays an important role in thermotolerance. During prolonged heat treatment, mutants of AtPLC3 showed decreased heat resistance. We observed no obvious phenotypic differences between plc3 mutants and wild type (WT) seedlings under normal growth conditions, but after heat shock, the plc3 seedlings displayed a decline in thermotolerance compared with WT, and also showed a 40-50% decrease in survival rate and chlorophyll contents. Expression of AtPLC3 in plc3 mutants rescued the heat-sensitive phenotype; the AtPLC3-overexpressing lines also exhibited much higher heat resistance than WT and vector-only controls. The double mutants of plc3 and plc9 displayed increased sensitivity to heat stress, compared with either single mutant. In transgenic lines containing a AtPLC3:GUS promoter fusion, GUS staining showed that AtPLC3 expresses in all tissues, except anthers and young root tips. Using the Ca(2+)-sensitive fluorescent probe Fluo-3/AM and aequorin reconstitution, we showed that plc3 mutants show a reduction in the heat-induced Ca(2+) increase. The expression of HSP genes (HSP18.2, HSP25.3, HSP70-1 and HSP83) was down-regulated in plc3 mutants and up-regulated in AtPLC3-overexpressing lines after heat shock. These results indicated that AtPLC3 also plays a role in thermotolerance in Arabidopsis, and that AtPLC3 and AtPLC9 function additionally to each other.

Cytokinin plays an important role in plant development and stress tolerance. Studies of Arabidopsis (Arabidopsis thaliana) have demonstrated that cytokinin acts through a two-component system that includes a histidine (His) kinase, a His phosphotransfer protein (HP), and a response regulator. Phylogenetic analyses have revealed the conservation of His kinases but lineage-specific expansion of HPs and response regulators in rice (Oryza sativa). However, whether the functions of rice HPs have diverged remains unknown. In this study, two rice authentic HPs (OsAHP1 and OsAHP2) were knocked down simultaneously via RNA interference (RNAi), and the transgenic OsAHP- RNAi plants exhibited phenotypes expected for a deficiency in cytokinin signaling, including dwarfism with reduced internode lengths, enhanced lateral root growth, early leaf senescence, and reduced tiller numbers and fertility under natural conditions. The OsAHP- RNAi seedlings were also hyposensitive to exogenous cytokinin. Furthermore, OsAHP- RNAi seedlings were hypersensitive to salt treatment but resistant to osmotic stress relative to wild-type plants. These results indicate that OsAHPs function as positive regulators of the cytokinin signaling pathway and play different roles in salt and drought tolerance in rice.

Traditionally, calmodulin (CaM) was thought to be a multi-functional receptor for intracellular Ca(2+) signals. But in the last ten years, it was found that CaM also exists and acts extracellularly in animal and plant cells to regulate many important physiological functions. Laboratory studies by the authors showed that extracellular CaM in plant cells can stimulate the proliferation of suspension cultured cell and protoplast; regulate pollen germination and pollen tube elongation, and stimulate the light-independent gene expression of Rubisco small subunit (rbcS). Furthermore, we defined the trans-membrane and intracellular signal transduction pathways for extracellular CaM by using a pollen system. The components in this pathway include heterotrimeric G-protein, phospholipase C, IP(3), calcium signal and protein phosphorylation etc. Based on our findings, we suggest that extracellular CaM is a polypeptide signal in plants. This idea strongly argues against the traditional concept that there is no intercellular polypeptide signal in plants.

The role of heterotrimeric G protein in signal transduction pathway of extracellular calmodulin in regulatingrbcS expression was examined in suspension-cultured cells of transgenic tobacco. Pharmalogical experiments indicated that G protein agonist cholera toxin enhancedrbcS expression and heterotrimeric G protein antagonist pertussis toxin inhibitedrbcS expression in transgenic tobacco cells. Pertussis toxin also inhibited the enhancement effect caused by exogenous purified calmodulin onrbcS expression, whereas cholera toxin completely reversed the inhibitory effects caused by anti-calmodulin serum onrbcS expression. The right side-out vesicles from tobacco cell membrane were purified, which contained all of substrates for fluometric assay of GTPase activity. Exogenous purified calmodulin, when adding directly to the medium of plasma membrane vesicles, significantly activated GTPase activity in the right side-out plasma membrane vesicles, and this increase in GTPase activity was completely inhibited both by heterotrimeric G proteins antagonist pertussis toxin and nonhydrolyzable GTP analogs GMP-PCP. These results provided the evidence that heterotrimeric G proteins may be involved in signal transduction pathways of extracellular calmodulin to regulaterbcS gene expression.

The peptide primary messengers regulate embryonic development, cell growth and many other activities in animal cells. But recent evidence verified that peptide primary messengers are also involved in plant defense responses, the recognition between pollen and stigma and keep the balance between cell proliferation and differentiations in shoot apical meristems. Those results suggest that plants may actually make wide use of peptide primary messengers, both in embryonic development and late life when they rally their cells to defend against pathogens and insect pests. The recent advance in those aspects is reviewed.