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Abstract: Two tobacco hydroxyproline-rich glycopeptide systemin precursor proteins (TobpreproHypSys-A and -B) have been discovered recently. The TobpreproHypSys-A, from which TobHypSys I and TobHypSys II were released, has been reported to be induced by mechanical and herbivorous wounding systemically. Here our experiments by overexpressing TobpreproHypSys-A in transgenic plants indicated the overexpression of TobpreproHypSys-A enhances the resistance against Helicoverpa armigera larvae. The growth and development of H. armigera larvae were reduced dramatically feeding on transgenic tobacco plants. To determine the causes of the increased resistance, both PIs and PPO as defense proteins were investigated by Northern blot and activity analysis. PIs expression and activity were dramatically induced in the TobpreproHypSys-A overexpressed plants. PPO activity was also induced substantially in transgenic plants. These data suggest that the enhanced resistance to insect larvae could result from the accumulation of PIs and PPO induced by the TobpreproHypSys-A expression in transgenic plants. [Copyright &y& Elsevier]

Many calmodulin-binding protein kinases have been isolated from plants. Plant calmodulin-binding protein kinases are novel protein kinases that differ from calcium-dependent protein kinases in many important respects. Calmodulin-binding protein kinases are likely to be crucial mediators of responses to diverse endogenous and environmental cues in plants. In this update, we review the structure, regulation, expression and possible functions of plant calmodulin-binding protein kinases. [Copyright &y& Elsevier]

As sessile organisms, plants require many flexible strategies to adapt to the environment. Although some environmental signaling pathways regulating stomatal development have been identified, how stomatal regulators are modulated by internal and external signals to determine the final stomatal abundance requires further exploration. In our studies, we found that nitric oxide (NO) promotes stomatal development with increased stomatal index as well as the relative number of meristemoids and guard mother cells [%(M+GMC)] in NO-treated wild-type Arabidopsis plants; this role of NO was further verified in the nox1 mutant, which exhibits higher NO levels, and the noa1 mutant, which exhibits low NO accumulation. To gain insight into the molecular mechanisms underlying the effect of NO, we further assayed the expression of genes involved in stomatal development and found that NO induces the expression of the master regulators SPCH , MUTE and SCRM2 to initiate stomatal development. In addition, MPK6 is also involved in NO-promoted stomatal development, as MPK6 expression was repressed in nox1 and NO-treated plants, and transgenic plants overexpressing MPK6 were less sensitive to SNP treatment in terms of changes in the%(M+GMC). Thus, our study shows that NO promotes the production of stomata by up-regulating the expression of SPCH , MUTE and SCRM2 and down-regulating MPK6 expression. [ABSTRACT FROM AUTHOR]

Abstract: A cDNA clone, encoding calcium (Ca2+)-dependent protein kinase (CDPK or CPK), was isolated from tobacco (Nicotiana tabacum). The full-length cDNA of 2360 bp contains an open reading frame for NtCPK4 consisting of 572 amino acid residues. Sequence alignment indicated that NtCPK4 shared high similarities with other CPKs and some CPK-related protein kinases (CRKs). Biochemical analyses showed that NtCPK4 phosphorylated itself and calf thymus histones fraction III-S (histone III-S) in a calcium-dependent manner, and the K 0.5 of calcium activation was 0.29 μM or 0.25 μM with histone III-S or syntide-2 as substrates, respectively. The V max and K m were 588 nmol min−1 mg−1 and 176 μg ml−1, respectively, when histone III-S was used as substrate, while they were 2415 nmol min−1 mg−1 and 58 μM, respectively, with syntide-2 as substrate. In addition, the phosphorylation of NtCPK4 occurred on threonine residue, as shown by capillary electrophoresis analyses. All of these data demonstrated that NtCPK4 was a serine/threonine protein kinase. NtCPK4 as a low copy gene was expressed in all tested organs including the root, leaf, stem, and flower of tobacco, while its expression was temporally and spatially modulated in both productive and vegetative tissues during tobacco growth and development. NtCPK4 expression was also increased in response to the treatment of gibberellin or NaCl. Our study suggested that NtCPK4 might play vital roles in plant development and responses to environmental stimuli. [Copyright &y& Elsevier]

• 14-3-3 κ and λ negatively regulate plant Cd tolerance. • 14-3-3 κ and λ decrease the accumulation of GSH and PC by modulating the synthesis gene expression under Cd stress. • 14-3-3 κ and λ function in plant Cd response by affecting H 2 O 2 content through changes of GSH accumulation. It is well-known that plants respond to the toxin cadmium (Cd), a widespread soil contaminant, by accumulating glutathione (GSH) and phytochelatin (PC). However, the factor(s) in the regulation of GSH and PC synthesis remain to be further identified. Here, we report that 14-3-3 κ and λ negatively regulate plant Cd tolerance because the double mutant 14-3-3κλ with higher Cd accumulation exhibited enhanced Cd tolerance, while overexpressing 14-3-3κ or 14-3-3λ resulted in increased Cd sensitivity in terms of changes of primary root elongation and fresh weight under Cd stress. The expression induction of the genes for GSH and PC synthesis and thus GSH and PC accumulation were further promoted in the 14-3-3κλ mutant but reduced in 14-3-3κ or 14-3-3λ overexpression lines compared with those in the stressed wild-type plants, suggesting the involvement of GSH and PC accumulation in 14-3-3s-mediated plant Cd response. This note was further supported by our observation that higher Cd tolerance was compromised in 14-3-3κλ seedlings treated simultaneously with Cd and buthionine sulfoximine, an inhibitor of GSH synthesis, compared with that in 14-3-3κλ seedlings treated with Cd alone. In addition, Cd-induced ROS accumulation was suppressed in 14-3-3κλ plants but further enhanced in 35S::14-3-3κ and 35S::14-3-3λ transgenic lines compared with that in wild-type plants, in consistent with GSH accumulations in these plants. Together, 14-3-3 s function in plant Cd response by changes of glutathione and glutathione synthesis in Arabidopsis. [ABSTRACT FROM AUTHOR]

With the development of industry, atmospheric SO 2 is rapidly increased, seriously affecting plant growth and crop production. However, the mechanism of how plants respond to high SO 2 -induced sulfite stress is poorly characterized. Here we report that ABA signaling is involved in plant tolerance to sulfite stress. Our results indicate that the sulfite-stressed plants accumulated higher ABA with promoted expression of ABA-responsive genes compared with unstressed control and the ABA-biosynthetic mutant ABA DEFICIENT2 (aba2–1) had less sulfite tolerance than wild-type plants, supporting a role of ABA in plant sulfite stress response. ABA functions through its downstream transcription factor ABF4 because the abf4–1 mutant was more sensitive but the 35 S::ABF4 plants more tolerant to the sulfite stress than wild-type plants. Further, ABF4 upregulates SOX expression by directly binding to its promoter and overexpression of SOX can rescue increased stress sensitivity of abf4–1 mutant, showing that SOX acts downstream of ABF4. Together, our study reveals that the sulfite-stressed plants can activate ABA/ABF4 signaling to enhance SOX expression in plant sulfite stress tolerance. • Sulfite stress induces the accumulation of ABA. • ABA protects plants from sulfite stress. • Sulfite stress promotes expression of transcription factor ABF4. • ABF4 increases plant sulfite stress tolerance by modulating SOX expression. [ABSTRACT FROM AUTHOR]

H 2 O 2 affects the expression of genes that are involved in plant responses to diverse environmental stresses; however, the underlying mechanisms remain elusive. Here, we demonstrate that H 2 O 2 enhances plant freezing tolerance through its effect on a protein product of low expression of osmotically responsive genes2 (LOS2). LOS2 is translated into a major product, cytosolic enolase2 (ENO2), and sometimes an alternative product, the transcription repressor c-Myc-binding protein (MBP-1). ENO2, but not MBP-1, promotes cold tolerance by binding the promoter of C-repeat/DRE binding factor1 (CBF1), a central transcription factor in plant cold signaling, thus activating its expression. Overexpression of CBF1 restores freezing sensitivity of a LOS2 loss-of-function mutant. Furthermore, cold-induced H 2 O 2 increases nuclear import and transcriptional binding activity of ENO2 by sulfenylating cysteine 408 and thereby promotes its oligomerization. Collectively, our results illustrate how H 2 O 2 activates plant cold responses by sulfenylating ENO2 and promoting its oligomerization, leading to enhanced nuclear translocation and transcriptional activation of CBF1. [Display omitted] • H 2 O 2 confers plant freezing tolerance under cold-acclimated conditions • ENO2 promotes plant cold response by binding to and activating CBF1 • H 2 O 2 sulfenylation at ENO2 Cys408 is involved in plant freezing tolerance • Cold-induced H 2 O 2 triggers ENO2 oligomerization and nuclear import Liu et al. examine the role of H 2 O 2 in the plant cold response. They find that H 2 O 2 , through sulfenylation that promotes the oligomerization of ENO2, enhances ENO2 nuclear translocation and transcriptional binding activity. ENO2 in turn upregulates the expression of the cold response regulator CBF1 , thus promoting freezing tolerance. [ABSTRACT FROM AUTHOR]

Although most ABC proteins are transporters, proteins of the ABC F subfamily lack the required transmembrane domains and, consequently, their functions are poorly understood. Here, we report that the F subfamily member general control non-repressible 20 (GCN20) mediates ABA and ROS accumulation in plants during salt stress. We demonstrate that salt conditions induce the expression of GCN20. The gcn20–1 mutant with more ABA accumulation is more sensitive to high salinity than the wild type in terms of decreased seed germination and fresh weight, and decreasing ABA accumulation can rescue reduced salt tolerance of the mutant, revealing the involvement of ABA in GCN20-mediated salt stress response. Consistently, salt stress promotes a number of stress-related and ABA signal-responsive genes expression in the gcn20–1 plants. In addition, the gcn20–1 plants have less CAT and SOD activity, and thus more ROS than wild-type plants when subjected to salt stress. These findings display that GCN20 is closely involved in the stress response caused by high salinity through modulation of ABA accumulation. • Salt stress promotes GCN20 expression in Arabidopsis. • GCN20 functions in plant response to salt stress response. • The sensitivity of gcn20-1 to salt stress involves ABA. • GCN20 modulates ROS homeostasis. [ABSTRACT FROM AUTHOR]

Abstract: Pollen deposition on stigmas and pollen tube growth in two apocarpous species, Ranalisma rostratum and Sagittaria guyanensis ssp. lappula (Alismataceae), were examined with fluorescence microscopy. The reallocation of pollen tubes among pistils was observed in both species. The percentage of pollinated stigmas per flower was only 22.0% in R. rostratum and 51.0% in S. guyanensis, though the seed/ovule ratios are higher than 65% in both species. The number of pollen grains on each single stigma ranged from 0 to 96 in R. rostratum, and from 0 to 125 in S. guyanensis. When more than one pollen grain deposited on a stigma, all pollen tubes grew to the ovary, but only one of them turned towards the ovule and finally entered the nucleus. The other tubes grew through the receptacle tissue into ovules of adjacent carpels whose stigmas were unpollinated or pollinated later. The intercarpellary growth of pollen tubes could be a mechanism to increase the efficiency of sexual reproduction in an apocarpous gynoecium with low pollination on the pistils. [Copyright &y& Elsevier]

Abstract: A cDNA clone from Arabidopsis corresponding to a typical CDPK-related protein kinase, AtCRK3, was isolated. AtCRK3 contains all conserved kinase domain, and has N-terminal myristoylation site and C-terminal domain containing apparently degenerate EF-hands. Biochemical analysis showed that the kinase activity of AtCRK3 for both autophosphorylation and substrate phosphorylation was neither enhanced by Ca2+, nor inhibited by EGTA, similar to other CRKs. In addition, the kinase activity of AtCRK3 is not stimulated by any of four Arabidopsis CaM isoforms, which is different from AtCRK1. Further experiments indicated that the V max and K m of AtCRK3 were 35.5nmolmin−1 mg−1 and 6.2μM, respectively when using histone IIIs as substrate. Capillary electrophoresis showed that autophosphorylation of AtCRK3 occurred on threonine residues. These data showed that AtCRK3 was a serine/threonine protein kinase. In situ hybridization showed that AtCRK3 was expressed in productive and vegetative tissues with temporal and spatial changes during Arabidopsis growth and development. This kinase was also induced in response to ABA treatment. Our study suggested that AtCRK3 might be an important signaling component in plant signaling transduction pathway. [Copyright &y& Elsevier]

A separation using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was applied to the study of green fluorescent protein tagged calmoldulin (GFP-CaM) that was expressed from Escherichia coli and purified with Ni2+-nitrilotriacetate (Ni-NTA) resin column. It was found that GFP-CaM not only has good fluorescence properties under various conditions similar to GFP, but also retains its calcium-binding ability as the native CaM. GFP-CaM was separated and detected by CE-LIF within 10 min with a limit-of-detection (LOD) of 2×10−10 M for an injection volume of 3 nl, higher than that of common chemical fluorescent-tagged protein method. The results indicated that, as a fluorescence probe, GFP could overcome the drawback of inefficient derivatization of chemical fluorescence probes. The interaction between the GFP-CaM and Ca2+ was studied in detail using affinity capillary electrophoresis with laser-induced fluorescence and the dissociation constant (Kd) between GFP-CaM and Ca2+ was determined to be 1.2×10−5 M, which is in good agreement with the literature values of untagged CaM (10−6 to 10−5 M) obtained by conventional method. As a preliminary application, the interaction between GFP-CaM and OsCBK was also investigated. The method makes it possible to screen the trace amounts of target proteins in crude extracts interacting with CaM under physiological conditions. [Copyright &y& Elsevier]

Calcium/calmodulin (CaM) plays vital roles in the regulation of many physiological and metabolic processes through its target proteins including calcium/CaM-modulated protein kinases. Here, we report our studies on the expression of a CaM-binding protein kinase gene (NtCBK2) during plant development and under some stimuli. Our results indicated that the expression of NtCBK2 was greatly enhanced after 10 min with high-salt or GA treatment, and reached the maximal at 30 min, while other stimuli, such as ABA, NAA, PEG-6000, cold (4 °C) or high temperature (42 °C) treatment had no effect on the expression of NtCBK2, suggesting possible involvement of NtCBK2 in salt and GA stress responses of plants. Using digoxigenin-labeled anti-sense RNA probe, the expression of NtCBK2 was detected in developing tobacco anther, pistil and embryo, revealing that the mRNA of NtCBK2 distributed mainly in actively dividing and vigorously metabolizing cells in a temporal and spatial manner during these developmental events. These data suggest that NtCBK2 may play a key role in the calcium/CaM-mediated signal pathway associated with floral development and embryogenesis in tobacco. [Copyright &y& Elsevier]

• Blue light induces adventitious root formation through the blue light receptors, PHOT1 and PHOT2. • The phototropic transducer NPH3 functions in blue light-induced adventitious root formation. • NPH3 physically interacts with PIN3. • NPH3 regulates adventitious root formation by affecting PIN3-mediated auxin transport. Adventitious rooting is a heritable quantitative trait that is influenced by multiple endogenous and exogenous factors in plants, and one important environmental factor required for efficient adventitious root formation is light signaling. However, the physiological significance and molecular mechanism of light underlying adventitious root formation are still largely unexplored. Here, we report that blue light-induced adventitious root formation is regulated by PIN-FORMED3 (PIN3)-mediated auxin transport in Arabidopsis. Adventitious root formation is significantly impaired in the loss-of-function mutants of the blue light receptors, PHOTOROPIN1 (PHOT1) and PHOTOROPIN2 (PHOT2), as well as the phototropic transducer, NON-PHOTOTROPIC HYPOCOTYL3 (NPH3). In addition, blue light enhanced the auxin content in the adventitious root, and the pin3 loss-of-function mutant had a reduced adventitious rooting response under blue light compared to the wild type. The PIN3 protein level was higher in plants treated with blue light than in those in darkness, especially in the hypocotyl pericycle, while PIN3-GFP failed to accumulate in nph3 PIN3::PIN3-GFP. Furthermore, the results showed that PIN3 physically interacted with NPH3, a key transducer in phototropic signaling. Taken together, our study demonstrates that blue light induces adventitious root formation through the phototropic signal transducer, NPH3, which regulates adventitious root formation by affecting PIN3-mediated auxin transport. [ABSTRACT FROM AUTHOR]