Plant Signaling & Behavior

Published by Taylor & Francis
Online ISSN: 1559-2324
Publications
High-resolution refraction-free DIC microscopy of tobacco pollen tube growth. (A) Pollen germination with exocytosis along the apical perimeter (arrow). (B) Pollen tube ca. 60 μm length with exocytosis in the subapical growth zone annulus (arrow). (C) Three successive growth cycles in a pollen tube undergoing oscillatory growth. Each row represents 1 cycle with elapsed time in s given above. Arrowheads mark surface features that appear before the start of the cycle. Exocytosis and cell elongation occur in the subapical growth zone marked with arrows. (D) Oscillatory growth in a lily pollen tube, elapsed time in s given above. Growth occurs in the structurally fluid subapical zone marked with arrow. (E and F) Correlation between growth rate oscillations and disruption of cell surface features resulting from deposition of new materials. Emergence of the growth zone annulus can occur immediately before (triple arrow) or just before (double arrow) the start of the growth cycle or at the end of the previous cycle (single arrow). (E) Tobacco pollen tube, shown in (C). (F) Lily pollen tube, shown in (D). Bars = 10 μm. 
Article
Our recent work used novel methods to localize and track discrete vesicle populations in pollen tubes undergoing oscillatory growth. The results show that clathrin-dependent endocytosis occurs along the shank of the pollen tube, smooth vesicle endocytosis occurs at the tip, and exocytosis occurs in the subapical region. Here, growth of tobacco and lily pollen tubes is examined in greater temporal resolution using refraction-free high-resolution time-lapse differential interference contrast microscopy. Images were collected at 0.21 s intervals for 10 min, sequentially examined for millisecond details, compressed into video format and then examined for details of growth dynamics. The subapical growth zone is structurally fluid, with vesicle insertion into the plasma membrane, construction of new cell surface and cellular expansion. Incorporation of new membrane and wall materials causes localized disruption at the cell surface that precedes the start of the growth cycle by 3.44 +/- 0.39 s in tobacco, and 1.02 +/- 0.01 s in lily pollen tubes. Vesicle deposition increases after the start of the growth cycle and supports expansion of the growth zone. Growth reorientation involves a shift in the position and angle of the growth zone. In summary, these results support a new model of pollen tube growth.
 
Article
Plasmodesmata (Pd), coaxial membranous channels that connect adjacent plant cells, are not static, but show a dynamic nature and can be opened or closed. These controlled changes in Pd conductivity regulate plant symplasmic permeability and play a role both in development and defense processes. One of the mechanisms shown to produce these changes is the deposition and hydrolysis of callose by beta-1-3-synthase and glucanase, respectively. Recently we have identified the first beta-1,3-glucanase Arabidopsis enzyme that is associated to the macromolecular Pd complex, termed AtBG_pap. When fused to GFP, this previously identified GPI-anchored protein localizes to the ER and the plasma membrane where it appears in a punctuate pattern that colocalizes with callose present around Pd. In T-DNA insertion mutants that do not transcribe AtBG_pap, GFP cell-to-cell movement between epidermal cells is reduced and callose levels around Pd are elevated. In this addenda we review the plant developmental processes of symplasmic regulation that have been shown to include callose deposition and beta-1,3-glucanase activity, and suggest a role for AtBG_pap in these processes. Additionally, based on the ability of viral movement proteins (MPs) to interact with ankyrin repeat proteins, and together with our recent findings showing the involvement of viral particles in callose degradation, we also purpose a new model for the ability of viruses to overcome Pd-callose deposition, and mediate their cell-to-cell movement.
 
Immunofluoresence labeling of callose by CH-II-512 antiserum (A, C and E) and b-1,3glucanases by P38-SH antiserum (B, D and F) on semi-thin serial sections of '474' embryogenic hybrid leaf fragments. (Bar = 20 mm). (A and B) In noninduced leaf fragments, small spots of callose are deposited on all the cell walls (A: arrows) and no b-1,3-glucanase is detected (B). (C and D) In three days induced leaves, callose and b-1,3-glucanases are detected on regions of a weak number of cell walls in the leaves (arrow heads). (E and F) In four days induced leaves, callose was labeled as deposits along the cell wall of a cell at the beginning of reactivation (large arrow head) and in cell walls of neighboring non reactivated cells (small arrow head). The reactivated cell (RC) is also labeled (arrow) (E) and b-1,3-glucanases are localized on the same cell walls (F). 
Article
During direct somatic embryogenesis in leaves of Cichorium hybrid clone '474', 38 kDa beta-1,3-glucanases are accumulated in the culture medium of the embryogenic hybrid to a higher level when compared with a non-embryogenic cultivar. In the same time, embryogenic cells were surrounded by a cell wall that was characterized by the presence of callose. This callosic deposition disappeared as embryos grew. Callose consisted of beta-1,3-glucan linkages and so represented a possible substrate for beta-1,3-glucanases. Using immunolocalization experiments, we demonstrated that from the three types of callose deposits observed during the culturing of Cichorium leaf explants, only the callose present in the walls surrounding reactivated cells seemed specifically related to somatic embryogenesis. Moreover, callose and the 38-kDa beta-1,3-glucanases were co-localized dispersed throughout the thick and swelled walls of reactivated cells and embryo cell walls. This suggests that callose and beta-1,3-glucanases are implicated in the process of somatic embryogenesis since they were always detected in or quite near embryogenic and embryo cell. This also suggested that beta-1,3-glucanases could be involved in the degradation of this callose.
 
Detection of the lectin ligand (urease) at the cell wall of Evernia phycobionts as a black deposit of CoS. (A) Control without urea and (B) reaction with urea of isolated algae from lichen thalli collected in November. (C) Control without urea and (D) with urea of algae isolated from lichen thalli collected in May. Bar = 5.0 µm. Figure 2. Loss of arginase activity after binding of the enzyme to the cell wall ligand. (A) binding to algal naturally containing the ligand (collected in November); (B) binding to algal cells containing the ligand after experimental induction by urea (collected in May). Black rectangles: total arginase added to cell suspension; white rectangles, arginase activity recovered in the supernatant after incubation with algal cells; grey rectangles; activity lost after binding to the ligand. Values are the mean of three replicates. Vertical bars give standard error.
Measurement of galactose released from Evernia phycobiont treated with α- and β-galactosidase
Binding of FITC-arginase to lichen phycobionts. (A) control without incubation with FITC-arginase; (B) phycobionts from thalli collected in November, treated with FITC-arginase and observed during irradiation with blue light; (C) phycobionts from thalli collected in November, previously incubated with urea and, after this, treated with FITC-arginase and observed during irradiation with blue light; (D) the same cells than in B incubated with α-galactosidase, then treated with FITC-arginase and observed during irradiation with blue light;E) the same cells than in (C) incubated with α-galactosidase, then treated with FITC-arginase and observed during irradiation with blue light; (F) the same cells than in (B) incubated with β-galactosidase, then treated with FITC-arginase and observed during irradiation with blue light; (G) the same cells than in (C) incubated with β-galactosidase, then treated with FITC-arginase and observed during irradiation with blue light; Bar = 5.0 µm.
Binding of FITC-labelled secretable arginase to the cell wall ligand of algae isolated from E. prunastri thalli collected in November. Values are the mean of three replicates. Bars give standard error.  
Article
A lectin from the lichen Evernia prunastri developing arginase activity (EC. 3.5.3.1) binds to the homologous algae that contain polygalactosilated urease (EC. 3.5.1.5) in their cell walls acting as a lectin ligand. The enzyme bound to its ligand shows to be inactive to hydrolyze of arginine. Hydrolysis of the galactoside moiety of urease in intact algae with alpha-1,4-galactosidase (EC. 3.2.1.22) releases high amount of D-galactose and impedes the binding of the lectin to the algal cell wall. However, the use of beta-,4-galactosidase (EC.3.2.1.23) releases low amounts of D-galactose from the algal cell wall and does not change the pattern of binding of the lectin to its ligand. The production of glycosilated urease is restricted to the season in which algal cells divide and this assures the recognition of new phycobiont produced after cell division by its fungal partner.
 
Article
Ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) is the most abundant protein on the planet and in addition to its central role in photosynthesis it is thought to function as a nitrogen (N)-storage protein and a potential source of N for defense biosynthesis in plants. In a recent study in the wild tobacco Nicotiana attenuata, we showed that the decrease in absolute N invested in soluble proteins and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis; (15)N flux studies revealed that N for defensive phenolamide synthesis originates from recently assimilated N rather than from RuBisCO turnover. Here we show that a transgenic line of N. attenuata silenced in the expression of RuBisCO (asRUB) invests similar or even larger amounts of N into phenolamide biosynthesis compared with wild type plants, consistent with our previous conclusion that recently assimilated N is channeled into phenolamide synthesis after elicitation. We suggest that the decrease in leaf proteins after simulated herbivory is a tolerance mechanism, rather than a consequence of N-demand for defense biosynthesis.
 
A model for the Vid pathway. When wild-type cells are starved of glucose for a prolonged period of time, significant amounts of FBPase are secreted into the periplasm. Following glucose re-feeding, FBPase is internalized into Vid/endosomes. The internalization of FBPase requires the SLA1, ARC18 and VPS34 genes. Under the same conditions, most of the Vid24p, Sec28p, Vid30p and Vps34p are in the intracellular fraction. Vid24p, Sec28p and Vid30p associate with actin patches initially and dissociate later, whereas Vps34p associates with actin patches constitutively. Following internalization, FBPase is targeted to the vacuole and then degraded in the lumen. 
Article
In Saccharomyces cerevisia, the key gluconeogenic enzyme fructose-1,6-bisphosphatase is secreted into the periplasm during prolonged glucose starvation and is internalized into Vid/endosomes following glucose re-feeding. Fructose-1,6-bisphosphatase does not contain signal sequences required for the classical secretory and endocytic pathways. Hence, the secretion and internalization are mediated via the non-classical pathways.
 
Article
Photoperiodic flowering in Arabidopsis is controlled not only by floral activators such as GI, CO, and FT, but also by repressors such as SVP and FLC. Double mutations in LHY and CCA1 (lhy;cca1) accelerated flowering under short days, mainly by the GI-CO dependent pathway. In contrast, lhy;cca1 showed delayed flowering under continuous light (LL), probably due to the GI-CO independent pathway. This late-flowering phenotype was suppressed by svp, flc, and elf3. However, how SVP, FLC, and ELF3 mediate LHY/CCA1 and flowering time is not fully understood. We found that lhy;cca1 exhibited short hypocotyls and petioles under LL, but the molecular mechanism for these effects has not been elucidated. To address these questions, we performed a screen for mutations that suppress either or both of the lhy;cca1 phenotypes under LL, using two different approaches. We identified two novel mutations, a dominant (del1) and a recessive (phyB-2511) allele of phyB. The flowering times of single mutants of three phyB alleles, hy3-1, del1, and phyB-2511, are almost the same and earlier than those of wild-type plants. A similar level of acceleration of flowering time was observed in all three phyB mutants tested when combined with the late-flowering mutations co-2 and SVPox. However, the effect of phyB-2511 on lhy;cca1 was different from those by hy3-1 or del1. svp-3 did not strongly enhance the early-flowering phenotypes of phyB-2511 or del1. These results suggest that light signaling via PhyB may affect factors downstream of the clock proteins, controlling flowering time and organ elongation. phyB mutations with different levels of effects on lhy;cca1-dependent late flowering would be useful to determine a specific role for PHYB in the flowering pathway controlled by lhy;cca1 under LL.
 
Article
In addition to its role in protein synthesis and the plant cells' response to environmental stresses, circumstantial evidence suggest that proline may also play a role in flowering and development both as a metabolite and as a signal molecule. Although there is a growing consensus that proline is of special importance throughout the reproductive phase (from flower transition to seed development) a general agreement on the molecular and genetic mechanisms proline is involved in, is yet to be established. In this paper we shall review and critically discuss most of the evidence supporting a role for proline in plant development, paying special attention to the recently reported role of proline in flower transition.
 
Article
Recently, we reported the genome-wide identification of 107 homeobox genes in rice and classified them into ten distinct subfamilies based upon their domain composition and phylogenetic analysis. Microarray analysis revealed the tissue-specific and overlapping expression profiles of these genes during various stages of floral transition, panicle development and seed set. Several homeobox genes were also found to be differentially expressed under abiotic stress conditions. Based on massively parallel signature sequencing (MPSS) data analysis, we report here that a large number of small RNA signatures are associated with rice homeobox genes, which may be involved in their tissue-specific/developmental regulation and stress responses. The association of a very large number of small RNA signatures suggested an unusually high degree of regulation of homeobox genes by small RNAs during inflorescence development.
 
Article
Polyamines have long been implicated in plant growth and development, as well as adaptation to abiotic and biotic stress. As a general rule of thumb the higher the polyamine titers the better. However, their molecular roles in plant stress responses still remain obscure. It has been postulated that they could act through their catabolism, which generates molecules which may act as secondary messengers signalling networks of numerous developmental and stress adaptation processes. Recently it was shown that plant and mammalian polyamine catabolism share critical features, giving new insight in plant polyamine catabolism. In this review, the advances in genes and proteins of polyamine catabolism in plants is presented and compared to other models.
 
Article
Heterotrimeric G-proteins play an important regulatory role in multiple physiological processes, including the plant immune response, and substantial progress has been made in elucidating the G-protein-mediated defense-signaling network. This mini-review discusses the importance of G-proteins in plant immunity. We also provide an overview of how G-proteins affect plant cell death and stomatal movement. Our recent studies demonstrated that G-proteins are involved in signal transduction and induction of stomatal closure and defense responses. We also discuss future directions for G-protein signaling studies involving plant immunity.
 
Article
RNA silencing inducible sequence (RSIS) causes post-transcriptional gene silencing (PTGS) of 5' or 3' flanking sequence-containing genes by inhibiting proper transcriptional termination. Exploiting this nature, 13 kD Pro-less, in which major seed storage protein (SSP) 13 kD prolamins are reduced, has been generated. In 13 kD Pro-less, other SSPs, such as glutelins, are increased as a compensation effect to maintain amino acid pool. 7Crp is the seven-linked epitope peptide derived from major cedar pollen allergens Cry j 1 and Cry j 2. When 7Crp is expressed in 13 kD Pro-less endosperm, accumulation level of 7Crp increased. Furthermore, recovery rate of 7Crp without reducing reagent increased. These findings indicate that 13 kD Pro-less endosperm provides a good production platform for recombinant proteins.
 
Article
The plant hormone ethylene (ET) plays a crucial role in the signalling network when plants have to respond to biotic stresses. We investigate the beneficial interaction between the model plant Arabidopsis thaliana and the endophytic fungus Piriformospora indica. Recently, we showed that ET signalling and ETHYLENE RESPONSE FACTOR (ERF)1 are important to balance beneficial and nonbeneficial traits in this symbiosis. 147 ERF genes in Arabidopsis encode transcriptional regulators with a variety of functions involved in development, physiological processes as well as plant/microbe interactions. In the beneficial symbiosis between Arabidopsis and P. indica, overexpression of ERF1 activates defence responses, strongly reduces root colonization and thus abolishes the benefits for the plants. Here we show that additional transcription factors of the ERF family, the ERF DOMAIN PROTEIN9 (ERF9) and the ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR14 (ERF14) are involved in the interaction between the two symbionts and are required for growth promotion of the host plant. Expression of these genes is upregulated in colonized wild-type roots. Insertional inactivation of ERF9 and ERF14 diminishes the P. indica-induced growth promotion and activates the expression of the PATHOGENESIS-RELATED (PR)-1 and PR-2 genes. We propose that ERF9 and ERF14 repress PR gene expression in colonized Arabidopsis roots and thus contribute to the establishment of a beneficial interaction.
 
Model for 14-3-3 structure and function. The fundamental structure of 14-3-3s is a conserved core (51% identity among Arabidopsis isoforms) that is flanked by highly divergent N and C terminal regions. The structure of the conserved central region is known to be a " w " -shaped clamp formed of two monomers, each capable of binding phosphorylated proteins or peptides. The complete structure and positions of the N and C termini (with reduced overall identity but with blocks of sequences that are conserved among evolutionary branches) are unresolved in the crystals, but the C termini likely form a movable flap that can seal the top of the clamp, perhaps depending upon divalent cation binding. The 14-3-3 shown is human Eta PDB:2C74 viewed with Cn3D v4.1 National Center for Biotechnology Information http://www/ncbi.nih.gov.  
Proteins in the photoperiodic flowering pathway. 14-3-3s are currently known to connect to photoperiodic flowering by interaction with CO and FT proteins, as indicated by the darkened symbols in the diagram. Some of the other proteins discussed in the text as being involved in flowering are shown in their approximate position in the pathways. Those within boxes have been tested for 14-3-3 interaction, but to date no interaction with these proteins and 14-3-3s has been reported.
Article
The 14-3-3 family of proteins is well known for participating in signal transduction by binding specifically phosphorylated proteins, thereby completing their kinase-induced transition in activity or localization. This interaction-based modulation of signal flux through metabolic pathways is a critical feature of many important eukaryotic signal transduction cascades. Only recently, however, have studies in Arabidopsis thaliana described that some of the most fundamental plant signal transduction pathways, including the photoperiodic flowering pathway, are functionally affected by 14-3-3s. There are pivotal points in the photoperiod pathway that are characterized by the accumulation, localization and stability of critical protein factors, all of which are strongly affected by light quality and photoperiod duration. These mechanisms (localization, phosphorylation, regulated proteolysis) are the same as those regulated by 14-3-3 proteins in other systems. Yet it is only recently that well characterized 14-3-3 genetic tools have become available in sufficient diversity to make it possible to truly tie 14-3-3 interactions to light signaling and flowering. This review presents an overview of photoperiodic flowering signaling and direct 14-3-3 participation in the process, coupled with a discussion of the overlapping and specific roles of 14-3-3s which present confounding issues in the functional dissection of this family of signaling proteins.
 
Article
Isoflavonoids are plant natural compounds predominantly found in leguminous plant. They play important functions in both nitrogen fixation and stress resistance. Many clinical studies have linked dietary intake of isoflavonoids to human health benefits. Binding of 14-3-3 proteins to GmMYB176, an isoflavonoid regulator, modulates expression of key isoflavonoids gene expression and its biosynthesis. We have recently demonstrated that the interaction of 14-3-3 proteins with GmMYB176 regulates nuclear-cytoplasmic localization of GmMYB176 thereby affecting target gene expression. Here, we report GmMYB62 as a new R1 MYB client protein of soybean 14-3-3s that may function together with GmMYB176 for gene regulation in soybean.
 
Article
Higher plants adapt to phosphorus deficiency through a complex of biological processes. Among of them, two adaptive processes are very important for the response of higher plants to phosphorus deficiency. One is the enhancement of root growth by regulating carbohydrate metabolism and allocation, and the other is rhizosphere acidification to acquire phosphorus efficiently from soil. TFT6 and TFT7, two different members of tomato 14-3-3 gene family, play the distinct roles in the adaption of plants to phosphorus deficiency by taking part in the two processes respectively. TFT6 which acts mainly in leaves is involved in the systemic response to phosphorus deficiency by regulating leaf carbon allocation and increasing phloem sucrose transport to promote root growth, while TFT7 directly functions in root by activating root plasma membrane H (+) -ATPase to release more protons under phosphorus deficiency. Based on these results, we propose that 14-3-3 proteins play the smart role in response to phosphorus deficiency in higher plants.
 
Article
We recently identified a novel R1 MYB transcription factor, GmMYB176, which regulates the CHS8 gene expression and influences isoflavonoid biosynthesis in soybeans. GmMYB176 recognizes a unique sequence motif [TAGT(T/A)(A/T)] in CHS8 promoter and binds with it. The in planta role of GmMYB176 was established by RNAi silencing of GmMYB176 in soybean hairy roots. Silencing of GmMYB176 reduced the expression of CHS8 gene expression and isoflavonoid accumulation in hairy roots. However, the overexpression of GmMYB176 did not lead to increase in both CHS8 expression and isoflavonoid level in hairy roots suggesting that GmMYB176 is essential but not sufficient for CHS8 gene activation.1.
 
Article
Abstract 14-3-3 proteins play essential roles in diverse cellular processes through the direct binding to target proteins. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in gibberellin (GA) feedback regulation. The 14-3-3 proteins bind to RSG depending on the RSG phosphorylation of Ser-114 and negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The Ca (2) (+)-dependent protein kinase NtCDPK1 was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of RSG. 14-3-3 weakly binds to NtCDPK1 by a new mode. The autophosphorylation of NtCDPK1 was necessary for the formation of the binding between NtCDPK1 and 14-3-3 but not for its maintenance. In this study, we showed that 14-3-3 binding to NtCDPK1 does not require the autophosphorylation when RSG was bound to NtCDPK1. These data suggest that 14-3-3 binds to an unphosphorylated motif in NtCDPK1 exposed by a conformational change in NtCDPK1 but not to a phosphate group generated by autophosphorylation of NtCDPK1.
 
Article
Members of the 14-3-3 family of proteins participate in signal transduction by modulating flux through various pathways. Potential subfunctionalization within this family has produced a suite of related proteins with diverse client interactions and discrete localization. The associated study assesses the biological roles of two specific 14-3-3 isoforms, using genetic, biochemical and physiological assays to ascertain potential nodes of interaction. Arabidopsis T-DNA insertion mutants representing the nu and mu isoforms exhibited a short, yet clear delay in flowering time on long days. Tests of hypocotyl growth inhibition under narrow bandwidth light indicated a hyposensitivity to red light, while responses to blue and far-red light were normal. These physiological tests suggest a mechanistic link between 14-3-3 proteins, red light sensing, and the pathways that control photoperiodic flowering. The precise entry point into the pathway was assessed using yeast two hybrid assays targeted against specific proteins active in the circadian oscillator, light transduction and photoperiodic flowering. Yeast two hybrid interaction was observed with CONSTANS (CO), and then confirmed with coimmunoprecipitation. Functional interaction with phyB leading to defects in flowering time and direct interaction with CONSTANS circumstantially places these specific 14-3-3 isoforms into the pathway that regulates the transition between vegetative and floral development.
 
Article
For a long time it has been believed that lignification has an important role in host defense against pathogen invasion. Recently, by using an RNAi gene-silencing assay we showed that monolignol biosynthesis plays a critical role in cell wall apposition (CWA)-mediated defense against powdery mildew fungus penetration into diploid wheat. Silencing monolignol genes led to super-susceptibility of wheat leaf tissues to an appropriate pathogen, Blumeria graminis f. sp. tritici (Bgt), and compromised penetration resistance to a non-appropriate pathogen, B. graminis f. sp. hordei. Autofluorescence of CWA regions was reduced significantly at the fungal penetration sites in silenced cells. Our work indicates an important role for monolignol biosynthetic genes in effective CWA formation against pathogen penetration. In this addendum, we show that silencing of monolignol genes also compromised penetration resistant to Bgt in a resistant wheat line. In addition, we discuss possible insights into how lignin biosynthesis contributes to host defense.
 
(A) Inoculation of pumpkin epicotyl with an Agrobacterium suspension. (B) Hairy root tumor growing within the lower stem of an A. rhizogenesinoculated plant. (C) Aspect of a tumor in a plant inoculated with A. tumefaciens. (D) Agarose gel electrophoresis of PCR products from different tissues from control and plants inoculated with A. rhizogenes harboring a GUS-GFP fusion gene under the control of the CaMV 35S promoter using GFP-specific primers. 1, positive control; 2, leaf from untransformed control plant; 3, hairy root tumor growing within the stem; 4, systemic leaf 5 cm from the apex; 5, root 8 cm from the inoculation site; 6; floral bud 1-2 cm from the apical meristem; 7, stem sample 8 cm from the inoculation site. (E) Agarose gel elctrophoresis of PCR products from different tissues from control and plants inoculated with A. tumefaciens using GFP-specific primers. Lanes 1-7, same as in D, except for 3, which is tumor from stem in the vicinity of the wounding site. 
CmPP16-overexpressing plants show enhanced photosynthetic activity relative to wild type controls and those expressing GFP-GUS. Shown here are plants transformed with A. tumefaciens harboring the CmPP16-GFP fusion construct. Photosynthesis (mmoles of assimilated CO 2 m ¡2 s ¡1 ) against days under drought treatment. FTSW was measured during this period and also plotted. 
Same as in Figure 3, except that the CmPP16-GFP construct was harbored by A. rhizogenes. 
Article
Abstract The phloem plays an important role in the delivery of nutrients and signals between photosynthetic to heterotrophic tissues. Proteins and RNAs in the phloem translocation stream may have an important role in maintaining the integrity of the sieve tube system, as well as in long-distance signaling. CmPP16 is a pumpkin phloem protein, which has been shown to bind RNA in a non-sequence specific manner, and move it cell-to-cell and conceivably, long-distance. The protein and RNA are found in both companion cell (CC) and sieve elements (SE). However, a more precise function for this protein is not known. In this work we report the overexpression of CmPP16 fused to GFP via transformation of pumpkin (Cucurbita maxima cv. Big Max) plants in the cotyledonary stage by direct inoculation of Agrobacterium tumefaciens and Agrobacterium rhizogenes. Plants overexpressing CmPP16 did not show an obvious phenotype. However, these plants displayed higher photosynthetic capacity during drought than wild-type (WT) pumpkin or transformed with another construct. These results suggest that CmPP16 may be involved in the response to stress through long-distance signaling.
 
Article
MicroRNAs (miRNAs) 165 and 166 are able to cleave their target mRNAs of HD-ZIP III genes, thus regulating the functions of these genes. Although it is generally accepted that both miR165 and miR166 perform the same functions in the regulation of HD-ZIP III genes in Arabidopsis, no experimental data are available to support this notion. Recent work has shown that overexpression of miR166 downregulates the expression of three HD-ZIP III genes, ATHB-9/PHV, ATHB-14/PHB and ATHB-15, which in turn recapitulates the phenotypes of simultaneous loss-of-function mutations of these genes. In the March issue of Plant & Cell Physiology, we have demonstrated that overexpression of miR165 leads to the down-regulation of all five HD-ZIP III genes, and concomitantly recapitulates the phenotypes of loss-of-function mutation of IFL1/REV and those of simultaneous loss-of-function mutations of IFL1/REV, ATHB-9/PHV and ATHB-14/PHB. These results indicate that miR165 and miR166 differentially regulate the functions of HD-ZIP III genes in Arabidopsis. In this addendum, we show that overexpression of the antisense form of the miR165a gene leads to formation of amphivasal vascular bundles, a phenotype reminiscent of that of the dominant mutation of IFL1/REV. This finding provides direct evidence for a role of miR165 in regulation of vascular patterning.
 
Article
Lyso-phosphatidylethanolamine (LPE) is a minor membrane glycerolipid and egg-derived 18:0-LPE is used commercially as a plant bio-regulator to improve plant product quality. Physiological responses initiated by LPE treatment included delayed senescence in leaves and fruits, improved shelf-life of products post harvest, and mitigation of ethylene-induced process. However, the biochemical and molecular mechanisms underlying LPE-induced responses in plants and harvested plant parts remain unclear. In this paper, commentary is presented on the effects of LPE at the biochemical level in an effort to develop a mode of action. Implications, although tentative, are that LPE exerts its effect via lipid-protein interaction to attenuate ethylene (ETH)-mediated responses and impact pathogenesis-related proteins which together delay senescence progression.
 
2,4-epibrassinolide induces lateral root (Lr) formation and inhibits primary root (Pr) elongation in a nitric oxide-dependent process in Arabidopsis. Col-0 Arabidopsis thaliana seedlings were grown vertically on atS plates for 5 d, treated later with 10 nm BL, 100 μm c-Ptio or 200 μm GSno. Seedlings were analyzed after 3 d of treatment. (A) representative images of the Arabidopsis seedlings. Bar: 1 cm. (B) For no detection, roots were incubated with 15 μm of the fluorescent probe DaF-FmDa and examined by epi-fluorescence (excitation 490 nm; emission 525 nm) in an Eclipse E 200 microscope (nikon). Bar: 75 min. (C) the ratio of Lr number/Pr length was taken as a measure of Lr density. Lr number only included those roots that were > 1 mm in length after 3 d of treatment. (D) Seedlings were photographed, and Pr length was measured using image J software (universal imaging). (E) For no quantification, DaF-Fm-Da fluorescence was analyzed with the image J 1.3 software and expressed as arbitrary units (a.u.). Values are the means ± SE of 5 independent experiments (n = 10). asterisks indicate significant differences at p < 0.05 (t-test). 
Characterization of no signaling operating downstream BL in Arabidopsis roots. Arabidopsis thaliana Col-0, nia1-2 and bri1-1 mutant lines were grown on atS plates for 5 d, treated later with 10 nm BL, 100 μm L-namE or 200 m GSno. Seedlings were analyzed after 3 d of treatment. (A) representative images of the Arabidopsis seedlings. Bar: 1 cm. (B) For no detection, roots were incubated with 15 μm of the fluorescent probe DaF-FmDa, and examined by epi-fluorescence. Bar: 75 min. Lr density (C), Pr length (D) and no (E) were quantified as indicated in Figure 1. Values are the means ± SE of five independent experiments (n = 10). asterisks indicate significant differences at p < 0.05 (t-test). 
Article
Brassinosteroids (BRs) regulate various physiological processes, such as tolerance to stresses and root growth. Recently, a connection was reported between BRs and nitric oxide (NO) in plant responses to abiotic stress. Here we present evidence supporting NO functions in BR signaling during root growth process. Arabidopsis seedlings treated with BR 24-epibrassinolide (BL) show increased lateral roots (LR) density, inhibition of primary root (PR) elongation and NO accumulation. Similar effects were observed adding the NO donor GSNO to BR-receptor mutant bri1-1. Furthermore, BL-induced responses in the root were abolished by the specific NO scavenger c-PTIO. The activities of nitrate reductase (NR) and nitric oxide synthase (NOS)-like, two NO generating enzymes were involved in BR signaling. These results demonstrate that BR increases the NO concentration in root cells, which is required for BR-induced changes in root architecture.
 
Scheme showing 2,4-D effect on leaf epinasty and cell death. 2,4-D induces ROS production. This ROS triggers leaf epinasty and oxidative processes giving rise senescence and cell death, but also participates as signal molecule activating defense cell response. ROS accumulation and gene expression are also regulated by changes in the hormone balance.
Article
Although structurally similar to the natural plant hormone indol-3- acetic acid, auxin herbicides were developed for purposes other than growth, and have been successfully used in agriculture for the last 60 years. Concerted efforts are being made to understand and decipher the precise mechanism of action of IAA and synthetic auxins. Innumerable results need to be interconnected to resolve the puzzle of auxin biology and action mode of auxin herbicides. To date, different breakthroughs are providing more insights into the process of plant-herbicide interactions. Here we highlight some of the latest findings on how the 2,4-dichlorophenoxyacetic acid damages susceptible broadleaf plants, emphasizing the role of ROS as a downstream component of the auxin signal transduction under herbicide treatment.
 
Article
Studies using inhibitors of indole-3-acetic acid (IAA) transport, not only for efflux but influx carriers, provide many aspects of auxin physiology in plants. 1-Naphtoxyacetic acid (1-NOA), an analog of the synthetic auxin 1-N-naphtalene acetic acid (NAA), inhibits the IAA influx carrier AUX1. However, 1-NOA also shows auxin activity because of its structural similarity to NAA. In this study, we have identified another candidate inhibitor of the IAA influx carrier. The compound, "7-B3; ethyl 2-[(2-chloro-4-nitrophenyl)thio]acetate," is a 2,4-dichlorophenoxyacetic acid (2,4-D) analog. At high concentrations (> 300 µM), 7-B3 slightly reduced IAA transport and tropic curvature of maize coleoptiles, whereas lower concentrations had almost no effect. We have analyzed the effects of 7-B3 on Arabidopsis thaliana seedlings. 7-B3 rescued the 2,4-D-inhibited root elongation, but not the NAA-inhibited root elongation. The effect of 7-B3 was weaker than that of 1-NOA. Both 1-NOA and 7-B3 inhibited DR5::GUS expression induced by IAA and 2,4-D, but not that induced by NAA. At high concentrations, 1-NOA exhibited auxin activity, but 7-B3 did not. Furthermore, 7-B3 inhibited apical hook formation in etiolated seedlings more effectively than did 1-NOA. These results indicate that 7-B3 is a potential inhibitor of IAA influx that has almost no effect on IAA efflux or auxin signaling.
 
Article
The correct title of the article in Plant Signaling & Behavior, Volume 5, Issue 7, 872-4: Role of nitric oxide and reactive oxygen species in disease resistance to necrotrophic pathogens. : Original article:: http://www.landesbioscience.com/journals/psb/article/11899/:
 
Article
The reference list for this article, originally published in V. 7, Issue 12 of Plant Signaling & Behavior, has been updated from the original version published online December 1st, 2012. The corrected PDF and Full-text versions of the article were posted Feb. 01, 2013 at the article webpage: http://www.landesbioscience.com/journals/psb/article/22471/
 
Model for the degradation of damaged proteins. (A) Stresses such as heat shock or the incorporation of amino acid analogues induce protein misfolding. If for example, a globular protein is misfolded, its hydrophobic core will be exposed to the cytoplasm. These hydrophobic regions can bind chaperones that either repair the misfolded protein or shuttle it to the ubiquitination enzymes and the 26SP. (B) Protein oxidation leads to a partial loss of secondary structure without disrupting the overall folding pattern of the protein, resulting in flexible, unstructured regions. These regions serve as degradation signals for the Ub-independent 20SP pathway. 
Article
Aggregation of proteins damaged by stress is often a causal factor of cell death. To prevent aggregation, eukaryotic cells rapidly degrade damaged proteins by engaging two types of proteasomes. The first type is the 26S proteasome (26SP) which is composed of a cylindrical proteolytic core-the 20S proteasome (20SP)-and one or two regulatory particles (RPs) that interact with ubiquitinated proteins. The second type is the free 20SP which mediates ubiquitin-independent proteolysis. We have recently shown that loss of RP function in Arabidopsis leads to an expected decrease in 26SP-dependent protein degradation and hypersensitivity to stresses that induce protein misfolding. Surprisingly, RP mutants have increased 20SP activity and tolerance to oxidative stress. This finding suggests that misfolded proteins carry one type of degradation signal that steers them to ubiquitination enzymes and the 26SP, while oxidatively damaged proteins carry another that guides them directly to the 20SP for degradation. Here we suggest that protein oxidation induces the formation of unstructured regions that serve as targeting signals for 20SP-dependent proteolysis.
 
Proteins and peptides identified by tandem mass spectrometry and database search 
Mass spectrometry analysis of sR45.1-GFP peptides. (A) sDs-PaGe showing the protein products from immunoprecipitation. (B) TOF m/z spectrum of peptides from (A). The 1816.7958 and 1816.7744 m/z peaks are for the (M+h) + ion of phosphorylated DGGPRRPReTsPQRK. Notice the absence of the ion for the non-phosphorylated analog which would theoretically be at 1737 m/z. (C) annotated tandem mass spectrum of phosphorylated DGGPRRPReTsPQRK. The observed m/z of the (M+h) + ion is 1816.83. The m/z for the phosphoric acid neutral loss ion is 1718.8 and is the dominant peak in the spectrum. (D) Protein domain structure showing the phosphorylation sites in sR45.1 identified by mass spectrometry. Phosphorylation sites, p1 from (C) and p2 identified in this study are indicated by arrows. Phosphorylation sites identified by previous studies 19,20 are indicated by *.
a summary of sR45-interacting proteins 
amino acid substitution tests for T 218. (A) The domain structure of sR45.1-GFP and residue substitution mutant constructs. (B) The GFP signal was examined in the nucleus of hypocotyl cells of 4-d-old seedlings as indicated. scale bars represent 10 μm. (C) Petal width-to-length (W/L) ratio of the col wild type, sr45-1 mutant and 2 independent lines for each transgene in the order as indicated in the figure. error bars present standard error of the mean (seM) from 40 flower petals. statistically significant differences (P < 0.001) are indicated by letters a-d. The GFP signal was also examined in trichomes of the sr45-1 mutant and each transgenic line. The same parameters (laser intensity = 80%, 488nm; Gain = 7.60B; Offset = 127) were used for all lines. scale bar = 25 μm.
alternative splicing pattern of SR30. (A) Two gene models, SR30.1 and SR30.5, showing an alternative 3′splice site within intron 10 in SR30.5. exons: black boxes; introns: straight lines; UTRs: white boxes; and alternative exon: gray box. stop codons in use are indicated by *. The position of primers used for RT-qPcR are shown as arrows. (B) The ratio of SR30.5/SR30.1 was examined in each genotype (n = 3). all ratios were compared with col. error bars present standard deviations. The student t-test was used for statistical analysis (* P < 0.05; ** P < 0.01).
Article
RNA splicing is crucial to the production of mature mRNAs (mRNA). In Arabidopsis thaliana, the protein Arginine/Serine-rich 45 (SR45) acts as an RNA splicing activator and initiates the spliceosome assembly. SR45 is alternatively spliced into two isoforms. Isoform 1 (SR45.1) plays an important role in the flower petal development whereas isoform 2 (SR45.2) is important for root growth. In this study, we used immunoprecipitation to isolate an SR45.1-GFP fusion protein from transgenic plants complementing a null mutant, sr45-1. Mass spectrometry suggested a single phosphorylation event in a peptide from the alternatively spliced region unique to SR45.1. Substituting alanine for threonine 218, a candidate site for phosphorylation, did not complement the sr45-1 mutant with narrow flower petals whereas substituting aspartic acid or glutamic acid for threonine 218 did complement the sr45-1 mutant. Mass spectrometry also revealed that other proteins involved in the spliceosome co-precipitated with SR45.1, and RT-qPCR revealed that phosphorylation of threonine 218 promotes the function of SR45.1 in promoting the constitutive splicing of SR30 mRNA. This is the first demonstration of a specific phosphorylation site that differentially regulates the function of a plant splicing activator in physiologically and morphologically distinct plant tissues.
 
Article
The transition metal copper is essential for all organisms yet excess copper is toxic because of production of free radicals via its free form. Therefore, the levels of copper are precisely regulated in a cell. Under copper depleted conditions, the expression of Cu/Zn superoxide dismutase (SOD) is downregulated and its function is compensated by Fe SOD in chloroplasts of higher plants. We presented evidence that a microRNA, miR398, is involved in this downregulation of Cu/Zn SOD genes in Arabidopsis thaliana when grown at low copper levels, corresponding to less than 1 microM Cu in tissue culture media. However, a green alga, Chlamydomonas reinhardtii, adjusts to copper depletion by modifying the photosynthetic apparatus from copper containing plastocyanin to iron containing cytochrome c(6). During evolution plants modified one of the main strategies to respond to copper deficiency probably to adapt to different metal environments.
 
(A) homology modeled structure for soybean CDPKβ with and without calcium built on the T. gondii CDPK structures (3hX4, plus Ca 2+ ; and 3Ku2, minus Ca 2+ ). two orientations, rotated 90°, are shown. the n-terminal domain is shown in red, the kinase domain in tan, the junction domain in magenta, and the Cam-like domain in purple. the horizontal dashed lines indicate the center placement of the kinase domains in the images. the tyr-24 structural region is indicated with the dashed black box, and is shown in expanded form in Figure 4. (B) the quality of the homology modeled CDPKβ structures assessed by QmEan is similar to other crystal structures. 
Article
Several plant CDPKs were recently shown to be dual specificity kinases rather than Ser/Thr kinases as traditionally classified by sequence analysis. In the present study we confirm the autophosphorylation of recombinant soybean His 6-GmCDPKβ at the Tyr-24 site using sequence- and modification- specific antibodies. Homology modeling of soybean CDPKβ based on recent structures determined for several apicomplexan CDPKs suggested that phosphotyrosine-24 may be inaccessible to phosphatases. However, we report that dephosphorylation of CDPKβ by the protein tyrosine phosphatase 1B, PTP1B, was not restricted in the presence of calcium. Thus, despite conformational changes likely associated with calcium binding to the CDPKs, phosphotyrosine sites remain fully accessible to dephosphorylation suggesting the possibility of conformational breathing and flexing.
 
Article
The 26S proteasome subunit RPT2 is a component of the hexameric ring of AAA-ATPases that forms the base of the 19S regulatory particle (RP). This subunit has specific roles in the yeast and mammalian proteasomes by helping promote assembly of the RP with the 20S core protease (CP) and gate the CP to prevent indiscriminate degradation of cytosolic and nuclear proteins. In plants, this subunit plays an important role in diverse processes that include shoot and root apical meristem maintenance, cell size regulation, trichome branching, and stress responses. Recently, we reported that mutants in RPT2 and several other RP subunits have reduced histone levels, suggesting that at least some of the pleiotropic phenotypes observed in these plants result from aberrant nucleosome assembly. Here, we expand our genetic analysis of RPT2 in Arabidopsis to shed additional light on the roles of the N- and C-terminal ends. We also present data showing that plants bearing mutations in RP subunit genes have their seedling phenotypes exacerbated by prolonged light exposure.
 
Mutant phenotypes suggest that the protein-level regulation is critical for normal leaf patterning. (A-E) Phenotypes of wild-type and single mutants. (A) wild-type Ler, (B) ae3-1, (C) ae5-1, (D) ae6-1 and (E) ae2-101. Insets in (B-D) show cauline leaves with an ectopic outgrowth (arrowheads) on their distal part of the abaxial side. (F-H) Double mutant phenotypes of ae3-1 as2-101 (F), ae5-1 as2-101 (G) and ae6-1 as2-101 (H). Arrowheads and arrows in (F-H) show the radially symmetric and lotus-like leaves with rough adaxial leaf surfaces, respectively. Bars = 5 mm in (A-H).
Article
Leaf morphogenesis requires the establishment of adaxial-abaxial polarity in emerging leaf primordia, and a number of genes participating in this process have been identified in recent years. We previously reported that the 26S proteasome is important in specifying the leaf adaxial fate. More recently, two papers from separate researches showed that several genes encoding ribosomal large subunit proteins also play an important role in leaf adaxial-abaxial patterning. Here we show that plants with a single mutation in the genes encoding either 26S proteasome subunits or ribosomal proteins shared similar abnormalities in some leaves, with an outgrowth formed on the distal part of the leaf abaxial side. Plants harboring these 26S proteasome or ribosome mutations in combination with an additional mutation asymmetric leaves1 or 2 (as1 or as2) demonstrated severely defective leaves, and the phenotypes of these double mutants were very similar. Because activities of the 26S proteasome and ribosome both affect the level of functional proteins, the recent findings suggest that a previously unrecognized regulation, the protein level regulation, is critical in normal leaf patterning. A regulatory model for the 26S proteasome and ribosome actions in leaf patterning is discussed.
 
Arabidopsis genes encoding chaperones involved in tubulin folding 
Article
In this review, we summarize the evidence pointing at the important role of 26S proteasome-dependent proteolysis in the regulation of microtubule synthesis and microtubule dynamics. Because most of the advances in this relatively unexplored research field originate from yeast and animal studies, we have considered those studies that describe the role of proteolysis in processes that are evolutionarily conserved and known to exist in plants. In addition, we place particular emphasis on the proteasome-dependent degradation of plant-specific microtubule-associated protein SPIRAL1 and its function in MT rearrangements associated with salt stress.
 
Article
The 26S proteasome interacts with a number of different proteins, while the TREX-2 complex is an important component of the mRNA export machinery. In animals and yeast, members of the Ubiquitin C-terminal Hydrolase 37 (UCH37) family are found to associate with the 26S proteasome, but this has not been demonstrated in plants. The Arabidopsis UCH1 and UCH2 are orthologous to UCH37. Here, we show that UCH1 and UCH2 interact with the 26S proteasome lid subunits. In addition, the two UCHs also interact with TREX-2 components. Our data suggest that Arabidopsis UCHs may serve as a link between the 26S proteasome lid complex and the TREX-2 complex.
 
Article
Leaf organogenesis occurs within the peripheral zone of the shoot apical meristem (SAM). It has been known that several members of the class-1 KNOTTED1-like homeobox (KNOX) genes are expressed in the SAM, and their expression must be prevented during leaf primordium initiation and subsequent leaf development. A number of regulators that repress class-1 KNOX genes have been identified, and characterizations of these regulators greatly improved our knowledge of the genetic basis of leaf organogenesis. We have recently reported that the proteolytic function of the Arabidopsis 26S proteasome is involved in specifying leaf adaxial identity during leaf development, by characterizations of mutants defective in genes encoding several 26S proteasome subunits. Here we demonstrate that in addition to the role in leaf polarity establishment, the 26S proteasome also participates in repression of class-1 KNOX genes during leaf development. We show that loss of functions in RPN8a and RPT2a, two 26S proteasome subunit genes, resulted in leaves that produce ectopic outgrowths on the abaxial side of blades. These outgrowths were accompanied by the ectopic expression of several class-1 KNOX genes. These results indicate that the 26S proteasome is important in repressing class-1 KNOX genes and its function may be required until later leaf developmental stages.
 
(A) Stability assay for tt8. two-week-old tt8-1seedlings expressing a 35Spro:tt8-FLaG (tt8-FLaGox) transgene were treated with 200 μm cycloheximide (ChX) for the indicated time period and used for Western blot analyses with anti-FLaG antibodies. Ponceau S-stained membrane with LSu is shown as a loading control. the relative amount of protein in each sample is normalized against the corresponding LSu. (B) Stabilization of tt8by mG132. two-week-old tt8-1 seedlings expressing a 35Spro:tt8-FLaG were treated with ChX, alone or in combination with mG132 (200 µm), for 120 minutes and used for Western blot analyses with anti-FLaG antibodies. DmSo-only treated plants served as control. the relative amount of protein in each sample is normalized against the corresponding LSu. (C) and (D) ttG1stability and mG132 stabilization assays were performed using two week-old ttg1 plants expressing a 35Spro:ttG1-FLaG (ttG1-FLaGox) as in (A) and (B). 
tt8-FLaG transcript levels in the seedlings, either untreated or treated with 200µm ChX, were examined using rt-PCr. Actin 2 was used as a control. tt8-FLaG transcript levels in the seedlings, treated with either DmSo or 200µm ChX (± 200µm mG132) for 120 minutes, were detected using rt-PCr. Actin 2 was used as a control. (A) and (D) ttG1 transcript levels were detected in 2 week-old ttg1 plants expressing35Spro:ttG1FLaG (ttG1-FLaGox) as in (A) and (B). 
Article
Regulated proteolysis by the ubiquitin/26S proteasome system (UPS) has emerged as a major posttranslational control mechanism regulating transcription factor (TF) activity in plants. Anthocyanin biosynthesis in Arabidopsis is regulated by a ternary complex comprised of basic helix-loop-helix (bHLH), R2R3MYB and WD-repeat (WDR) proteins. The bHLH TF, TRANSPARENT TESTA 8 (TT8), and the WDR protein, TRANSPARENT TESTA GLABRA1 (TTG1), are essential for expression of late flavonoid biosynthesis genes. Previous studies have demonstrated that the turnover of several anthocyanin pathway regulators is controlled by the UPS. Here, we showthatTT8 and TTG1areshort-lived and targeted by the UPS for degradation. Our findings further extend our understanding of the role of the UPS in the regulation of anthocyanin biosynthesis in plants.
 
Article
The enzyme HMG-CoA reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis, critical not only for normal plant development, but also for the adaptation to demanding environmental conditions. Consistent with this notion, plant HMGR is modulated by many diverse endogenous signals and external stimuli. Protein phosphatase 2A (PP2A) is involved in auxin, abscisic acid, ethylene and brassinosteroid signaling and now emerges as a positive and negative multilevel regulator of plant HMGR, both during normal growth and in response to a variety of stress conditions. The interaction with HMGR is mediated by B" regulatory subunits of PP2A, which are also calcium binding proteins. The new discoveries uncover the potential of PP2A to integrate developmental and calcium-mediated environmental signals in the control of plant HMGR.
 
Article
Controlled protein dephosphorylation by protein phosphatase 2A (PP2A) regulates diverse signaling events in plants. Recently, we showed that a specific B’γ regulatory subunit of PP2A mediates basal repression of immune reactions in Arabidopsis thaliana. Knock-down pp2a-b’γ mutants display constitutive defense reactions and premature yellowing conditionally under moderate light intensity. Here we show that knock-down of PP2A-B’γ renders CALRETI CULIN 1 (CRT 1) highly phosphorylated. Calreticulins are ER-resident chaperonins that operate in the unfolded protein response to prevent ER-stress, components of which are differentially regulated at mRNA level in pp2a-b’γ leaves. We speculate that in dephosphorylated state, CRT 1 promotes the degradation of unfolded proteins in ER. Our findings suggest that in wild type plants, dephosphorylation of CRT 1 is mediated by PP2A-B’γ dependent signaling effects. In pp2a-b’γ, strong phosphorylation of CRT 1 may partially imbalance the quality control of protein folding, thereby eliciting ER-stress and premature yellowing in leaves.
 
Article
Canonical protein phosphatase 2A (PP2A) consists of a catalytic subunit (C), a scaffolding subunit (A), and a regulatory subunit (B). The B subunits are believed to confer substrate specificity and cellular localization to the PP2A complex, and are generally divided into three non-related families in plants, i.e., B55, B' and B''. The two Arabidopsis B55 subunits (α and β) interact with nitrate reductase (NR) in the bimolecular fluorescence complementation assay in planta, and are necessary for rapid activation of NR. Interestingly, knockout of all four B55 alleles is probably lethal, because a homozygous double knockout (pp2a-b55αβ) could not be found. The B55 subunits, therefore, appear to have essential functions that cannot be replaced by other regulatory B subunits. A double mutant (pp2a-b'αβ) of two close B' homologs show severely impaired fertility, pointing to the essential role also of B' subunits in plant development.
 
Article
Serine/threonine protein phosphatases are ubiquitous enzymes in all eukaryotes but many of their physiological roles in plants remain unknown. The available results have demonstrated critical functions for these enzymes in the regulation of adaptive stress responses, and recent studies have directed attention to the functional roles of Ser/Thr phosphatases type 2A (PP2A) as components of stress signaling pathways. This review is focused primarily on plant PP2As and their participation in the control of biotic and abiotic stress responses.
 
Article
The 2b protein encoded by Cucumber mosaic virus (CMV) and other cucumoviruses is multifunctional, having roles in local and systemic virus movement, symptom determination, evasion of defense mediated by salicylic acid, and in suppression of antiviral RNA silencing. It also perturbs silencing-mediated regulation of host transcripts, suggesting that another function of 2b protein is to manipulate host gene expression and physiology in a way that may aid the virus. The 2b proteins encoded by the various cucumoviruses (CMV strains, as well as Tomato aspermy virus and Peanut stunt virus) share conserved amino acid sequence domains, suggesting that these might determine specific functions of the protein. We analyzed the effect of mutations in these domains on functions of the 2b protein during viral infection. This revealed that binding of short RNAs, the key determinants of RNA silencing specificity, correlates with RNA silencing suppression activity. Two putative phosphorylation sites were found to be required for virus symptom induction, despite having no influence on RNA silencing suppression. This indicates that the ability to suppress silencing is not the only factor affecting symptom induction by the 2b protein. In accordance with this, our studies also revealed that the 2b protein acts synergistically with some other CMV product(s) to induce symptoms, and that the role of the 2b protein in symptom determination is host species specific.
 
Article
Plant viral symptoms are rarely explained by direct molecular interaction between a viral protein and a host factor, but rather understood as a consequence of arms race between host RNA silencing and viral silencing suppressors. However, we have recently demonstrated that the 2b protein (2b) of Cucumber mosaic virus (CMV) HL strain could bind to Arabidopsis catalase that is important in scavenging cellular hydrogen peroxide, leading to the induction of distinct necrosis on Arabidopsis. Because we previously used virulent strains of subgroup I CMV in the study, we here further analyzed mild strains of subgroup II CMV, which share 70 to 80% sequence homology with subgroup I, to understand whether the necrosis induction is a general phenomenon to compromise host defense system mediated by catalase in the pathosystem of any CMV strains and Arabidopsis. Based on the results, we concluded that 2bs of subgroup II could also bind to catalase, resulting in decrease in catalase activity and weak necrosis on Arabidopsis. Because the 2b-catalase interaction did not prevent CMVs from spreading, it may eventually operate in favor of CMV.
 
qPCR of G. max roots overexpressing defense-related genes. qPCR was performed using primers designed specifically against LSD1–2 The experiments used the ribosomal S21 gene 8 as a control to standardize the experiments qPCR using LSD1 primers 
Representative control and transgenic LSD1-2 overexpressing and LSD1-2 RNAi G. max plants. (A) Control susceptible G. max [Williams 82/PtdIns 518671] plant. (B) Genetically engineered G. max [Williams 82/PI 518671] overexpressing Gm-LSD1-2. (C) Control resistant G. max [Peking/PtdIns 548402] plant. (D) A resistant G. max [Peking/PI 548402] plant genetically engineered to express an LSD1-2 RNAi construct. Scale provided on left of each image.
The female index for transgenic G. max plants genetically engineered to overexpress Gm-LSD1-2 and infected with H. glycines. Replicate 1 (R1) control plants had 28.39 cysts per gram (12 plants); LSD1-2R1-overexpressing plants (LSD1-2-R1: oe) had 13.66 cysts per gram (12 plants). The FI D 47.92; P-value D 0.0216541 which is statistically significant (P < 0.05). R2 control plants (replicate 2) had 30.40 cysts per gram (16 plants); LSD1-2-R2-overexpressing plants (LSD1-2-R2: oe) had 9.85 cysts per gram (12 plants). The FI D 32.4; P-value D 0.000059234 which is statistically significant (P < 0.05). R3 control plants had 32.98 cysts per gram (20 plants); LSD1-2-R3 overexpressing plants (LSD1-2-R3: oe) had 14.07 cysts per gram (18 plants). The FI D 42.662; P-value D 3.36219e-06 which is statistically significant (P < 0.05).
G. max plants genetically engineered for RNAi of Gm-LSD1-2 and infected with H. glycines have an increased capability, shown as fold change, for parasitism. Replicate 1 (R1) control plants (resistant G. max [Peking/PtdIns 548402] ) had 1.98 cysts per gram (10 plants). LSD1-2-RNAi-R1 (LSD1-2-R1: RNAi) in resistant G. max [Peking/PI 548402] ) had 6.41 cysts per gram (11 plants). The results were statistically significant (p D 0.00255251). Replicate 2 (R2) control plants (resistant G. max [Peking/PtdIns 548402] ) had 0.79 cysts per gram (12 plants). LSD1-2-RNAi-R2 (LSD1-2-R2: RNAi) in resistant G. max [Peking/PI 548402] ) had 8.63 cysts per gram (5 plants). The results were statistically significant (p D 0.0117053). Replicate 3 (R3) control plants (resistant G. max [Peking/PtdIns 548402] ) had 2.51 cysts per gram (10 plants). LSD1-2-RNAi-R3 (LSD1-2-R3: RNAi) in resistant G. max [Peking/PI 548402] ) had 11.7 cysts per gram (7 plants). The results were statistically significant (p D 0.0120138).
Article
Abstract Experiments show the membrane fusion genes alpha soluble NSF attachment protein (α-SNAP) and syntaxin 31 (Gm-SYP38) contribute to the ability of Glycine max to defend itself from infection by the plant parasitic nematode Heterodera glycines. Accompanying their expression is the transcriptional activation of the defense genes ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and NONEXPRESSOR OF PR1 (NPR1) that function in salicylic acid (SA) signaling. These results implicate the added involvement of the antiapoptotic, environmental response gene LESION SIMULATING DISEASE1 (LSD1) in defense. Roots engineered to overexpress the G. max defense genes Gm-α-SNAP, SYP38, EDS1, NPR1, BOTRYTIS INDUCED KINASE1 (BIK1) and xyloglucan endotransglycosylase/hydrolase (XTH) in the susceptible genotype G. max[Williams 82/PI 518671] have induced Gm-LSD1 (Gm-LSD1-2) transcriptional activity. In reciprocal experiments, roots engineered to overexpress Gm-LSD1-2 in the susceptible genotype G. max[Williams 82/PI 518671] have induced levels of SYP38, EDS1, NPR1, BIK1 and XTH, but not α-SNAP prior to infection. In tests examining the role of Gm-LSD1-2 in defense, its overexpression results in ∼52 to 68% reduction in nematode parasitism. In contrast, RNA interference (RNAi) of Gm-LSD1-2 in the resistant genotype G. max[Peking/PI 548402] results in an 3.24-10.42 fold increased ability of H. glycines to parasitize. The results identify that Gm-LSD1-2 functions in the defense response of G. max to H. glycines parasitism. It is proposed that LSD1, as an antiapoptotic protein, may establish an environment whereby the protected, living plant cell could secrete materials in the vicinity of the parasitizing nematode to disarm it. After the targeted incapacitation of the nematode the parasitized cell succumbs to its targeted demise as the infected root region is becoming fortified.
 
influence of the as-1 element of the camV 35S promoter on PR-1 1294 activity in wild-type and tga256 mutant plants. (a) Sequences of the as-1-like element of the PR-1 promoter and the as-1 element of the camV 35S promoter. conserved positions within the tGa binding site tGac/Gtca (gray box) are depicted in boldface letters; each tGac half site is marked by an arrow provided that 3 or 4 bps are in consensus with this motif. Vertical lines denote the centers of the palindromes. Bases that are different in the camV 35S-derived as-1 element as compared to the PR-1 as-1-like element are represented as lowercase letters. (B) luciferase activities of 2-week-old plants grown axenically on plates without (gray columns) and with 30 μm ina (black columns) were measured. the genotypes of the analyzed plants are indicated. Values are the means of the activities of the indicated number (#) of independent lines. luciferase activities are expressed as relative light units (rlu) per μg of total protein. error bars represent the standard deviation of two independent experiments. experimental conditions are as previously reported in reference 15. wt, wild-type.
influence of the as-1 element of the camV 35S promoter on PR-1 816-573 activity in wild-type, npr1 and sni1 plants. (a) Schematic presentation of the PR-1 816-573 promoter construct. Sequences between bp positions -573 and -68 and upstream of bp position -816 were removed. the black box depicts the relative position of the as-1-like element. (B) luciferase activities of 2 week-old plants grown axenically on plates without (gray columns) and with 30 μm ina (black columns) were measured. the genotypes of the analyzed plants are indicated. Values are the means of the activities of the indicated number (#) of independent lines. luciferase activities are expressed as relative light units (rlu) per μg of total protein. error bars represent the standard deviation of two independent experiments. experimental conditions are as previously reported in reference 15. wt, wild-type.
Article
The plant defense hormone salicylic acid (SA) activates gene expression through a number of different mechanisms. In Arabidopsis thaliana, the SA-induced PATHOGENESIS RELATED (PR)-1 promoter is regulated through TGA transcription factors binding to the two TGACG motifs of the so called as-1 (activation sequence-1)-like element which is located between base pair positions -665 and -641. Activation is mediated by the transcriptional co-activator NPR1 (NON EXPRESSOR OF PR GENES1), which physically interacts with TGA factors. Moreover, the promoter is under the control of the negative regulator SNI1 (SUPPRESSOR OF NPR1, INDUCIBLE1). We have recently reported that SNI1-mediated repression of basal promoter activities and NPR1-dependent induction are maintained in a truncated PR-1 promoter that contains sequences between -816 and -573 upstream of the -68 promoter region. In this addendum, we report that the expression characteristics of this truncated PR-1 promoter is changed profoundly when its as-1-like element is replaced by the as-1 element of Cauliflower Mosaic Virus 35S promoter which also contains two TGACG motifs. The resulting chimeric promoter showed high constitutive activity that was independent from SA, NPR1 and SNI1. Thus, the configuration of two TGA binding sites within the PR-1 promoter determines whether NPR1 can induce and whether SNI1 can repress the promoter.
 
Confocal images showing GFP-talin expression in 35S-GT and HGT lines. (A-C) Merged confocal images displaying auto fluorescence of chloroplasts (in red) and fluorescence signal of GFP (in green). (A) 35S-GT grown under standard conditions. Ten days old, dark grown caulonemal cells of (B) 35S-GT and (C) HGT lines. The 35S-GT cells were imaged immediately after light exposure, whereas HGT cells were heat treated in darkness for 1 h at 36°C and imaged 16 hours after treatment. Bars: 100 μm. 
35S-Hm line is unable to grow in the dark when hygromycin is supplemented to the medium. Growth of WT (left), 35S-Hm (middle) and Act-Hm (right) lines using mediums supplemented with (A and C) or without (B and D) hygromycin (Hm, 25 mg/l). Six days old P. patens colonies were transferred to dark (A and B) or continued to grow in standard growing conditions (C and D) during ten days. Pictures were taken immediately after. Bars: 10 mm. 
Article
The constitutive Cauliflower Mosaic Virus 35S promoter (CaMV 35S) is widely used as a tool to express recombinant proteins in plants, but with different success. We previously showed that the expression of an F-actin marker, GFP-talin, in Physcomitrella patens using the CaMV 35S promoter failed to homogenously label moss tissues. Here, we show a significant diminution of the GFP fluorescence in dark grown old moss cells and complete lack of labelling in newly differentiated cells. Furthermore, we demonstrate that stable moss lines harbouring a resistance cassette driven by the CaMV 35S are unable to grow in darkness in the presence of the antibiotic. In contrast to the CaMV 35S, the heat inducible promoter, hsp17.3B showed uniform expression pattern in all cells and tissues following a mild heat shock.
 
Article
The concept of plant intelligence, as proposed by Anthony Trewavas, has raised considerable discussion. However, plant intelligence remains loosely defined; often it is either perceived as practically synonymous to Darwinian fitness, or reduced to a mere decorative metaphor. A more strict view can be taken, emphasizing necessary prerequisites such as memory and learning, which requires clarifying the definition of memory itself. To qualify as memories, traces of past events have to be not only stored, but also actively accessed. We propose a criterion for eliminating false candidates of possible plant intelligence phenomena in this stricter sense: an "intelligent" behavior must involve a component that can be approximated by a plausible algorithmic model involving recourse to stored information about past states of the individual or its environment. Re-evaluation of previously presented examples of plant intelligence shows that only some of them pass our test.
 
Lateral root and root hair phenotype of wild-type (Wt) and nal2/3 plants with exogenous auxin treatment. Surface-sterilized rice seeds were germinated and grown on murashige and Skoog (mS) agar medium for 1 or 2 d. then, germinated seeds were transferred to mS agar plates containing 0.1 μm iaa (+iaa), or to control plates (-iaa) and grown vertically in the growth chamber. two-week-old plants were observed. (a-h) Lateral roots and root hair phenotypes of Wt and nal2/3 seedlings grown on +iaa (a-D) or -iaa (E-h) agar plates. White asterisks indicate emerging lateral roots. Lr, lateral root; Pr, primary root; rh, root hair. Bars = 2 mm in (a-D) and 0.3 mm in (E-h).  
tentative model for the coordinated regulation of lateral root and root hair development by osWoX3a-mediated auxin distribution in Wt and nal2/3. altered expression of OsPIN genes compromises the establishment of auxin maxima in pericycle cells (black arrowheads) where lateral roots initiate, but increases auxin concentration in the epidermal cells (white arrowheads), leading to reduction of lateral root initiation and increase of root hair initiation.  
Article
Coordinated regulation of the many genes controlling leaf, flower, and root development determines the phenotypes of plants; this regulation requires exquisite control of many transcription factors, including the WUSCHEL-related homeobox (WOX) family. We recently reported that rice (Oryza sativa) WUSCHEL-related homeobox 3A (OsWOX3A) plays important roles in organ development, including lateral-axis outgrowth and vasculature patterning in leaves, lemma and palea morphogenesis in spikelets, and the numbers of tillers and lateral roots. OsWOX3A is encoded by NARROW LEAF2 (NAL2) and NAL3, a pair of duplicated genes. In this study, further analysis of nal2 nal3 (hereafter nal2/3) double mutants revealed that, in addition to its role in lateral root development, OsWOX3A also acts in the control of root hair formation. Based on this new finding, we describe a possible mechanism by which OsWOX3A regulation of auxin transport genes acts in root development.
 
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Article
Abstract The recently developed transparent soil consists of particles of Nafion, a polymer with a low refractive index (RI), which is prepared by milling and chemical treatment for use as a soil analogue. After the addition of a RI-matched solution, confocal imaging can be carried out in vivo and without destructive sampling. In a previous study, we showed that the new substrate provides a good approximation of plant growth conditions found in natural soils. In this paper, we present further development of the techniques for detailed quantitative analysis of images of root-microbe interactions in situ. Using this system it was possible for the first time to analyse bacterial distribution along the roots and in the bulk substrate in vivo. These findings indicate that the coupling of transparent soil with light microscopy is an important advance towards the discovery of the mechanisms of microbial colonisation of the rhizosphere.
 
Top-cited authors
Narendra Tuteja
  • International Centre for Genetic Engineering and Biotechnology
Ramakrishna Akula
  • Monsanto Holdings Private Limited
Ravishankar Gokare
  • Dayananda Sagar Institutions
Sarvajeet Singh Gill
  • Maharshi Dayanand University
Michael Gabriel Paulraj
  • Guru Nanak College