ZmMS39 encodes a callose synthase essential for male fertility in maize(Zea mays L.)

Callose contributes to many biological processes of higher plants including pollen development,cell plate and vascular tissue formation,as well as regulating the transport function of plasmodesmata.The functions of callose synthase genes in maize have been little studied.We describe a maize male-sterile mutant 39(ms39)characterized by reduced plant height.In this study,we confirmed using CRISPR/Cas9 technology that a mutation in Zm00001d043909(ZmCals12),encoding a callose synthase,is responsible for the male sterility of the ms39 mutant.Compared with male-fertile plants,callose deposition around the dyads and tetrads in ms39 anthers was significantly reduced.Increased cell autophagy observed in ms39 anthers may have been due to the premature programmed cell death of tapetal cells,leading to collapse of the anther wall structure.Disordered glucose metabolism in ms39 may have intensified autophagy in anthers.Evaluation of the ms39 gene on maize heterosis by paired-crossed experiment with 11 maize inbred lines indicated that ms39 can be used for maize hybrid seed production.

Transient expression of the <i>Arabidopsis thaliana</i>callose synthase PMR4 increases penetration resistance to powdery mildew in barley

Localized cell wall thickenings, so called papillae, are a common plant defense response to fungal attack at sites of penetration of the plant cell. The major constituent of papillae is callose, a (1,3)-β-glucan polymer, which contributes to slowing or blocking the invading fungal hyphae. In the model plant Arabidopsis thaliana, we could recently show that the overexpression of PMR4(POWDERY MILDEW RESITANT 4), which encodes a stress induced callose synthase, results in complete powdery mildew resistance. To evaluate if these findings are also transferable to monocot crops, we transiently expressed PMR4 under control of the 35S promoter in leaves of barley (Hordeum vulgare) seedlings, which were subsequently inoculated with the virulent powdery mildew Blumeria graminis f. sp. hordei. Fusion of the green fluorescent protein (GFP) to PMR4 allowed the identification of successfully transformed barley cells, which showed an increased penetration resistance to B. graminis compared to control cells that express only GFP.PMR4-GFP localized in a similar pattern at the site of attempted fungal penetration as observed inA. thaliana, which suggests that similar transport mechanisms of the callose synthase might exist in dicot and monocot plants.

H_(2)O_(2)mediates transcriptome reprogramming during Soybean mosaic virus-induced callose deposition in soybean

The main defense response to Soybean mosaic virus(SMV)infection in soybean[Glycine max(L.)Merr.]is thought to be blockage of intercellular virus transport by callose deposition on plasmodesmata.But the specific regulatory mechanism remains largely unknown.In this study,we found that hydrogen peroxide(H_(2)O_(2))signal downstream of NO was associated with the regulation of callose accumulation.Abundant H_(2)O_(2)was produced on the cell membrane and cell wall in the incompatible combination of soybean cultivar Jidou 7 and SMV strain N3,whereas no obvious H_(2)O_(2)was observed in the compatible combination of Jidou 7 and strain SC-8.When H_(2)O_(2)production was inhibited,callose accumulation induced by SMV infection decreased to a level insufficient to restrict virus transport in the incompatible combination.The H_(2)O_(2)-associated transcriptome dynamics of soybean during SMV infection was investigated.Transcriptome and functional analysis using virus-induced gene silencing showed that Gm SEOB and Gm PAP27,two genes regulated by H_(2)O_(2),functioned in resistance by positively regulating the accumulation of callose in response to SMV infection.These results lay a foundation for further research on the signal transduction and molecular regulation of callose deposition during soybean resistance to SMV infection.

Defective callose walls and cell plates during abnormal meiosis cause male-sterility in the oat mutant zbs1

During meiosis in flowering plants,degradation of the callose wall in tetrads releases newly produced microspores,which develop into mature pollen grains.In this study,we identified zbs1,a male-sterile mutant of naked oat（Avena nuda L.）that displayed complete spikelet sterility due to inviable mature pollen.The abnormal pollen grains originated from microspores with a defective callose wall and cell plate during meiosis.The defective callose wall and cell plate of the zbs1 mutant were detected by the labeling of cell wall epitopes（β-1,3-glucan） with immunogold during meiosis,and an abnormal chromosome configuration was observed by propidium iodide staining.The mature pollen grains of the zbs1 mutant were irregular in shape,and abnormal germination was observed by scanning electron microscopy.Together,our results indicate that the cause of male sterility in zbs1 is abnormal meiosis.

The IBI1 Receptor ofβ-Aminobutyric Acid Interacts with VOZ Transcription Factors to Regulate Abscisic Acid Signaling and Callose-Associated Defense

Extemal and internal signals can prime the plant immune system for a faster and/or stronger response to pathogen attack.β-aminobutyric acid(BABA)is an endogenous stress metabolite that induces broad-spectrum disease resistance in plants.BABA perception in Arabidopsis is mediated by the aspartyl tRNA synthetase IBI1,which activates priming of multiple immune responses,including callose-associated cell wall defenses that are under control by abscisic acid(ABA).However,the immediate signaling components after BABA perception by IBI1,as well as the regulatory role of ABA therein,remain unknown.Here,we have studied the early signaling events controlling IBI1-dependent BABA-induced resistance(BABAIR),using untargeted transcriptome and protein interaction analyses.Transcriptome analysis revealed that IBI1-dependent expression of BABA-IR against the biotrophic oomycete Hyaloperonospora arabidopsidis is associated with suppression of ABA-inducible abiotic stress genes.Protein interaction studies identified the VOZ1 and VOZ2 transcription factors(TFs)as IBI1-interacting partners,which are transcrip-tionally induced by ABA but suppress pathogen-induced expression of ABA-dependent genes.Furthermore,we show that VOZ TFs require nuclear localization for their contribution to BABA-IR by mediating augmented expression of callose-associated defense.Collectively,our study indicates that the IBI1-VOZ signaling module channels pathogen-induced ABA signaling toward cell wall defense while simultaneously suppressing abiotic stress-responsive genes.

Delayed callose degradation restores the fertility of multiple P/TGMS lines in Arabidopsis

Photoperiod/temperature-sensitive genic male sterility(P/TGMS)is widely applied for improving crop production.Previous investigations using the reversible male sterile(rvms)mutant showed that slow development is a general mechanism for restoring fertility to P/TGMS lines in Arabidopsis.In this work,we isolated a restorer of rvms–2(res3),as the male sterility of rvms–2 was rescued by res3.Phenotype analysis and molecular cloning show that a point mutation in UPEX1 l in res3 leads to delayed secretion of callase A6 from the tapetum to the locule and tetrad callose wall degradation.Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis demonstrated that the tapetal transcription factor ABORTED MICROSPORES directly regulates UPEX1 expression,revealing a pathway for tapetum secretory function.Early degradation of the callose wall in the transgenic line eliminated the fertility restoration effect of res3.The fertility of multiple known P/TGMS lines with pollen wall defects was also restored by res3.We propose that the remnant callose wall may broadly compensate for the pollen wall defects of P/TGMS lines by providing protection for pollen formation.A cellular mechanism is proposed to explain how slow development restores the fertility of P/TGMS lines in Arabidopsis.

Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro

The conditions that favor the in vitro synthesis of cellulose from tobacco BY-2 cell extracts were determined. The procedure leading to the highest yield of cellulose consisted of incubating digitonin extracts of membranes from 11-day-old tobacco BY-2 cells in the presence of 1 mM UDP-glucose, 8 mM Ca^2＋ and 8 mM Mg^2＋. Under these conditions, up to nearly 40% of the polysaccharides synthesized in vitro corresponded to cellulose, the other polymer synthesized being callose. Transmission electron microscopy analysis revealed the occurrence of two types of structures in the synthetic reactions. The first type consisted of small aggregates with a diameter between 3 and 5 nm that associated to form fibrillar strings of a maximum length of 400 nm. These structures were sensitive to the acetic/nitric acid treatment of Updegraff and corresponded to callose. The second type of structures was resistant to the Updegraff reagent and corresponded to straight cellulose microfibrils of 2-3 nm in diameter and 200 nm to up to 5 μm in length. In vitro reactions performed on electron microscopy grids indicated that the minimal rate of microfibril elongation in vitro is 120 nm/min. Measurements of retardance by liquid crystal polarization microscopy as a function of time showed that small groups of microfibrils increased in retardance by up to 0.047 nm/min per pixel, confirming the formation of organized structures.

Comparative analysis of Wanjincheng orange leaf and root responses to ‘Candidatus liberibacter asiaticus’ infection using leaf-disc grafting

Citrus Huanglongbing,associated with Candidatus Liberibacter asiaticus(Las),is the serious disease of citrus worldwide.Here,we compared differences between leaf and root responses in Wanjincheng Orange(Citrus sinensis Osbeck)to Las infection using leaf-disc grafting.Trees had no obvious symptoms in the first two months after grafting(MAG),but yellowing leaves,thickened midribs and decayed roots began to appear at 6 MAG.The Las growth rate in roots was greater than inmidribs from 2 to 6 MAG;however,by 10 MAG,it was significantly lower in roots than in midribs.Amicroscopic analysis revealed that starch accumulation,callose deposition,cellwall thickness and the number of cell layers increased in the phloem of infected trees.After Las infection,the starch content in the leaves was significantly higher than that in the roots,while the callose deposition in the midribs was 3–19 times that in the roots.A gene expression analysis showed that transcripts of callose-and starch metabolism-related genes were obviously affected by Las infection,and the pathogenesis-related genes PR1,PR2 and PR5 were significantly upregulated and downregulated in midrib and root,respectively.Our results indicated that the PR-mediated resistance response was repressed in root but activated in midrib,which may result in the more rapid Las growth in roots than in midribs during the early infection stage.

Potassium sulphate induces resistance of rice against the rootknot nematode Meloidogyne graminicola

Potassium(K),an important nutrient element,can improve the stress resistance/tolerance of crops.The application of K in resisting plant-parasitic nematodes shows that the K treatment can reduce the occurrence of nematode diseases and increase crop yield.However,data on K_(2)SO_(4)induced rice resistance against the root-knot nematode Meloidogyne graminicola are still lacking.In this work,K_(2)SO_(4)treatment reduced galls and nematodes in rice plants and delayed the development of nematodes.Rather than affecting the attractiveness of roots to nematodes and the morphological phenotype of giant cells at feeding sites,such an effect is achieved by rapidly priming hydrogen peroxide(H_(2)O_(2))accumulation and increasing callose deposition.Meanwhile,galls and nematodes in rice roots were more in the potassium channel OsAKT11 and transporter OsHAK5 gene-deficient plants than in wild-type,while the K_(2)SO_(4)-induced resistance showed weaker in the defective plants.In addition,during the process of nematode infection,the expression of jasmonic acid(JA)/ethylene(ET)/brassinolide(BR)signaling pathway-related genes and pathogenesis-related(PR)genes OsPR1 a/OsPR1 b was up-regulated in rice after K_(2)SO_(4)treatment.In conclusion,K_(2)SO_(4)induced rice resistance against M.graminicola.The mechanism of inducing resistance was to prime the basal defense and required the participation of the K^(+)channel and transporter in rice.These laid a foundation for further study on the mechanism of rice defense against nematodes and the rational use of potassium fertilizer on improving rice resistance against nematodes in the field.

Hyperhidrosis of Central Hypogonadism Leading to Diagnosis of Shapiro Syndrome: Temperature Dysregulation, Hypothermia and Agenesis of the Corpus Callosum

Background: Central or hypothalamic hypogonadism as an initial manifestation of Shapiro Syndrome has not been described in the literature. Herein, we report first case in which initial presentation of central hypogonadism led to a confirmed diagnosis of Shapiro Syndrome during a casual evaluation of hypothalamic pituitary anatomy with MRI of brain. Case presentation: 53 year old Caucasian man was documented to manifest Central or hypogonadotropic hypogonadism following evaluation of excessive sweating episodes, lack of libido and erectile dysfunction for a duration of several years. Brain MRI performed for assessment of the etiology documented no pituitary abnormality. Instead agenesis of Corpus Callosum was noted. The subject had been hospitalized on many occasions at this and several other medical centers with hypothermia or hyperthermia without a documentation of a definite cause. Therefore, the diagnosis of Shapiro Syndrome was made. Conclusion: This report is the first documentation of subject manifesting central, more likely to be hypothalamic rather than hypogonadotropic hypogonadism in conjunction with Shapiro Syndrome.

Riboflavin-induced Priming for Pathogen Defense in Arabidopsis thaliana

Riboflavin （vitamin B2） participates in a variety of redox processes that affect plant defense responses. Previously we have shown that riboflavin induces pathogen resistance in the absence of hypersensitive cell death （HCD） in plants. Herein, we report that riboflavin induces priming of defense responses in Arabidopsis thaliana toward infection by virulent Pseudomonas syringae pv. tomato DC3000 （Pst）. Induced resistance was mechanistically connected with the expression of defense response genes and cellular defense events, including H202 burst, HCD, and callose deposition in the plant. Riboflavin treatment and inoculation of plants with Pst were neither active but both synergized to induce defense responses. The priming process needed NPRI （essential regulator of systemic acquired resistance） and maintenance of H202 burst but was independent of salicylic acid, jasmonic acid, ethylene, and abscisic acid. Our results suggest that the role of riboflavin in priming defenses is subject to a signaling process distinct from the known pathways of hormone signal transduction.

Cytological and Molecular Characteristics of Pollen Abortion in Lily with Dysplastic Tapetum

Lily was grown worldwide as a fresh cutting flower because of its colorful petals, but its anther contained a large number of pollen grains that cause serious pollen contamination, however, pollen abortion can effectively reduce the level of pollen pollution. Our analysis aims to use cytological observation to detect the critical stage when pollen abortion occurs and to provide comprehensive gene expression information at the transcriptional level. The result showed that pollen abortion in ‘Little Kiss’ began at the mononuclear stage and the callose that covers the microspores failed to degenerate when young pollens were released from the tetrads. In addition, compared with the normally developed one,the tapetum of ‘Little Kiss’ degraded in advance while the degradation of callose was delayed. Furthermore, 103 differentially expressed genes(DEGs) related to the advance degeneration of tapetum cells and callose were found in the expression levels, including 22 transcription factors(TFs). In particular, two β-glucanase genes(endo-1,3(4)-β-glucanase, exo-β-glucanase) responsible for callose degeneration were significantly down-regulated. These results suggested that pollen abortion may occur at mononuclear stage and that early degeneration of tapetum cells resulted in a significant down-regulation of β-glucanase genes. As a result, the callose to cover microspores impedes the formation of pollen walls, which may possibly lead to pollen abortion.

Evidence for the Role Evolutionary Increase Yield in Cotton of Transfer Cells in the in Seed and Fiber Biomass

Transfer cells （TCs） are specialized cells exhibiting invaginated wall ingrowths （Wls）, thereby amplifying their plasma membrane surface area （PMSA） and hence the capacity to transport nutrients. However, it remains unknown as to whether TCs play a role in biomass yield increase during evolution or domestication. Here, we examine this issue from a comparative evolutionary perspective. The cultivated tetraploid AD genome species of cotton and its A and D genome diploid progenitors displayed high, medium, and low seed and fiber biomass yield, respectively. In all three species, cells of the innermost layer of the seed coat juxtaposed to the filial tissues trans-differentiated to a TC morphology. Electron microscopic analyses revealed that these TCs are characterized by sequential formation of flange and reticulate Wls during the phase of rapid increase in seed biomass. Significantly, TCs from the tetraploid species developed substantially more flange and reticulate Wls and exhibited a higher degree of reticulate WI formation than their progenitors. Consequently, the estimated PMSA of TCs of the tetraploid species was about 4 and 70 times higher than that of TCs of the A and D genome progenitors, respectively, which correlates positively with seed and fiber biomass yield. Further, TCs with extensive Wls in the tetraploid species had much stronger expression of sucrose synthase, a key enzyme involved in TC Wl formation and function, than those from the A and D progenitors. The analyses provide a set of novel evidence that the development of TC Wls may play an important role in the increase of seed and fiber biomass yield through polyploidization during evolution.

Integrating Hormone- and Micromolecule- Mediated Signaling with Plasmodesmal Communication

Intercellular and supracellular communications through plasmodesmata are involved in vital processes for plant development and physiological responses. Micro- and macromolecules, including hormones, RNA, and proteins, serve as biological information vectors that traffic through the plasmodesmata between cells. Previous studies demonstrated that the plasmodesmata are elaborately regulated, whereby a long queue of multiple signaling molecules forms. However, the mechanism by which these signals are coupled or coor- dinated in terms of simultaneous transport in a single channel remains a puzzle. In the last few years, several phytohormones that could function as both non-cell-autonomous signals and plasmodesmal regulators have been disclosed. Plasmodesmal regulators such as auxin, salicylic acid, reactive oxygen species, gibberellic acids, chitin, and jasmonic acid could regulate intercellular trafficking by adjusting plasmodesmal permeability. Here, callose, along with β-glucan synthase and β-glucanase, plays a critical role in regulating plasmodesmal permeability. Interestingly, most of the previously identified regulators are capable of diffusing through the plasmodesmata. Given the small sizes of these molecules, the plasmo- desmata are prominent intercellular channels that allow diffusion-based movement of those signaling molecules. Obviously, intercellular communication is under the control of a major mechanism, named a feedback loop, at the plasmodesmata, which mediates complicated biological behaviors. Prospective research on the mechanism of coupling micromolecules at the plasmodesmata for developmental signaling and nutrient provision will help us to understand how plants coordinate their development and photosynthetic assimilation, which is important for agriculture.

Identification and Preliminary Characterization of a New Chemical Affecting Glucosyltransferase Activities Involved in Plant Cell Wall Biosynthesis

Chemical genetics as a part of chemical genomics is a powerful and fast developing approach to dissect biological processes that may be difficult to characterize using conventional genetics because of gene redundancy or lethality and, in the case of polysaccharide biosynthesis, plant flexibility. Polysaccharide synthetic enzymes are located in two main compartments--the Golgi apparatus and plasma membrane-and can be studied in vitro using membrane fractions. Here, we first developed a high-throughput assay that allowed the screening of a library of chemicals with a potential effect on glycosyltransferase activities. Out of the 4800 chemicals screened for their effect on Golgi glucosyltransferases, 66 compounds from the primary screen had an effect on carbohydrate biosynthesis. Ten of these compounds were confirmed to inhibit glucose incorporation after a second screen. One compound exhibiting a strong inhibition effect （ID 6240780 named chemical A） was selected and further studied. It reversibly inhibits the transfer of glucose from UDP-glucose by Golgi membranes, but activates the plasma membrane-bound callose synthase. The inhibition effect is dependent on the chemical structure of the compound, which does not affect endomembrane morphology of the plant cells, but causes changes in cell wall composition. Chemical A represents a novel drug with a great potential for the study of the mechanisms of Golgi and plasma membrane-bound glucosyltransferases.

Aphid populations showing differential levels of virulence on Capsicum accessions

The green peach aphid,Myzus persicae,is one of the most threatening pests in pepper cultivation and growers would benefit from resistant varietices.Previously,we identified two Capsicum acessions as susceptible and three as resistant to M.persicae using an aphid population originating from the Netherlands(NL).Later on we identified an aphid population originating from a diferent gcographical region(Switserland,SW)that was virulent on all tested Capsicum acessions.The objeetive of the current work is to describe in detail diferent aspects of the interaction between two aphid populations and two sclected Capsicum acessions(one that was susceptible[PB2013046]and one that was resistant[PB2013071]to population NL),including biochemical processes involved.Electrical penetration graph(EPG)recordings showed similar feeding activities for both aphid populations on PB2013046.On acession PB2013071 the aphid population sw was able to devote significantly more time to phloem ingestion than population NL.We also studied plant defense response and found that plants of acession PB2013046 could not induce an accumulation of reactive oxygen species and callose formation after infestation with either aphid population.However,plants of PB2013071 induced a stronger defense response after infestation by population NL than after infestation by population SW.Based on these results,population SW of M.persicae seems to have overcome the resistance of PB2013071 that prevented feeding of aphids from NL population.The potential mechanism by which SW population overcomes the resistance is discussed.