Optimization of Two-Dimensional Gel Electrophoresis for Kenaf Leaf Proteins

To establish a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis （2-DE） analysis in kenaf leaf tissues, three extraction methods （trichloroacetic acid/acetone, urea/thiourea, and phenol extraction methods） were applied to the extraction of kenaf leaf protein. The results were compared in regard to protein extraction efficiency, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and 2-DE gels. Furthermore, the 2-DE system was optimized for four aspects： the pH range of IPG （immobilized pH gradient） stripes, sampling methods, sample volumes, and concentration of polyacrylamide gels. The data presented showed that the phenol extraction method is the best method to perform 2-DE analysis of kenaf leaf protein. The protein extracted from phenol extraction method reached the purity of （26.40±0.859）%, showed （25.67±1.53） protein bands in one dimension SDS-PAGE gels, and （1 374±54.44） protein spots on 2-DE gels. The research also indicates that kenaf leaf protein spots were distributed mainly within the pH range of 4-8. More clear background with a better distribution effect and many protein spots could be obtained on 2-DE gels under the conditions of active rehydration loading, 24 cm IPG strips （linear pH gradient of 4-7）, 1.4 mg samples, and 12% SDS-PAGE gels.

Evaluation of seed production in a first-generation seed orchard of Chinese pine (Pinus tabuliformis)

We evaluated seed production in a first-gener- ation orchard of Chinese pine （Pinus tabuliformis） during the crucial transition period from first generation to advanced generations. Clones varied significantly in all traits related to seed production. Repeatability of these traits ranged from 0 to 0.96. Seed production per ramet （SPPR）, seed producing index （SPI）, the number of relative female strobili （RFS）, the number of scales, and the num- ber of ineffective scales had comparatively high repeata- bility at 0.86, 0.87, 0.89, 0.96, and 0.91, respectively. Correlation analysis showed that SPPR was greatly influenced by RFS and by the number of full seeds per cone. Finally, we showed that SPI was the best predictor of the seed-producing ability of clones. Our findings will assist seed orchard managers in effectively predicting and improving seed production of Chinese pine seed orchards.

Successful Agrobacterium-mediated transformation of Populus tomentosa with apple SPDS gene

The problem of salinized soils has become one of the most serious constraints to agricultural and forest productivity. With the purpose of enhancing salt stress tolerance of Populus tomentosa, we transformed this tree species with spermidine synthase （SPDS） genes derived from an apple by an Agrobacterium-mediated method. Four transgenic clones were confu＇med by PCR and Southern blot analysis. As well, the expression of introduced SPDS genes was analyzed by real-time quantitative PCR.

Environmental contribution to needle variation among natural populations of Pinus tabuliformis

Variations in the phenotypic characteristics of conifer needles is a consequence of genetic evolution that has been widely used in geographic variation and ecological studies.Although many studies are based on an in situ sampling strategy and generally realize the contribution of environmental effects to variation in needle traits,it is still uncertain which needle traits are most influenced by genetic effects and which are most influenced by the environment.Using both a common garden experiment to eliminate environmental heterogeneity and an in situ sampling strategy,we compared 18 Pinus tabuliformis needle traits among 10 geographical populations.Using both sampling strategies,we found significant differences in needle traits among populations and among individuals within populations.Differences in the‘‘among-population’’variance component between the two sampling strategies revealed the environmental contribution among natural populations for each trait.The among-population variance in the following traits exceeded 8%:needle length,number of stomata within 2 mm(NS2),number of stomatal lines on the planar side,number of resin canals(RCN)and the resin canal area(RCA).For the stability of needle traits,NS2,RCN,RCA,ratio of the vascular bundle area to the RCA(VBA/RCA),and MA/RCA differed significantly in more than five provenance changes between the common garden populations and natural populations,which may be susceptible to environmental effects.Conversely,the crosssectional area,mesophyll area(MA),MA/(VBA+RCA),and MA/VBA were phenotypically stable.Geographic variation patterns and systematic relation of needle traits differed between the two sampling strategies,suggesting that in situ sampling results may reflect environmental effects and deviate statistical parameters for genetic study.Future studies of genetic evolution in the context of geographic variation should be based on appropriate sampling strategies and stable phenotypic traits.

Interspecific Variation and Phylogenic Architecture of Pinus densata and the Hybrid of Pinus tabuliformis×Pinus Yunnanensis in the Pinus densata Habitat:an Electrical Impedance Spectra Perspective

We evaluated a novel and non-destructive method of the electrical impedance spectroscopy(EIS)to elucidate the genetic and evolutionary relationship of homoploid hybrid conifer of Pinus densata(P.d)and its parental species Pinus tabuliformis(P.t)and Pinus yunnanensis(P.y),as well as the artificial hybrids of the P.t and P.y.Field common garden tests of96 trees sampled from 760 seedlings and 480 EIS records of 1,440 needles assessed the interspecific variation of the P.d,P.t,P.y and the artificial hybrids.We found that(1)EIS at different frequencies diverged significantly among germplasms;P.y was the highest,P.t was the lowest,and their artificial hybrids were within the range of P.t and P.y;(2)maternal species effect of EIS magnitudes in the hybrids and P.d was stronger than the paternal species characteristics;(3)EIS of the artificial hybrid confirmed the mid-parent and partial maternal species characteristics;(4)unified exponential model of EIS for the interspecific and hybrids can be constructed as|Z|=Af^(-B);(5)cluster analysis for species and hybrid combinations in total corroborated with the previous hybrid model of Pinus densata.Our non-destructive EIS method complemented the previous finding that Pinus densata was originated from P.t and P.y.We conclude that the impedance would be a viable indicator to investigate the interspecific genetic variations of conifers.

Effect of Cadmium Stress on the Growth, Antioxidative Enzymes and Lipid Peroxidation in Two Kenaf (Hibiscus cannabinus L.) Plant Seedlings

The effects of cadmium stress on the growth, antioxidative enzymes and lipid peroxidation in two kenaf plants, Fuhong 991 and ZM412, were analysed under control （0.5-strength Hoagland＇s nutrient solution） or five levels of cadmium stress （0.5- strength Hoagland＇s nutrient solution containing different concentrations of Cd2＋）. The leaves and roots of control and cadmium-stressed plants were harvested after 3 wk. At the same Cd concentration, the Cd tolerance index of Fuhong 991 was higher than that of ZM412, indicating that Fuhong 991 may be more tolerant to Cd than ZM412. The superoxide dismutase （SOD）, catalase activity （CAT） and peroxidase （POD） activities fluctuated in the leaves of the Cd-stressed plants compared to the control, whereas the glutathione reductase activity （GR） was much larger than the control for Fuhong 991, ensuring that sufficient quantities of GSH were available to respond to the cadmium stress. In comparison to the control, the dynamic tendency of the SOD, CAT and POD activities in roots of the Cd-stressed plants all increased and then declined, but the POD activity of Fuhong 991 remained nearly unchanged at all of the stress levels. The increase in the enzyme activities demonstrated that Fuhong 991 was more tolerant to cadmium than ZM 412. The lipid peroxidation was enhanced only in the leaves of Cd-stressed ZM 412. These findings indicated that antioxidative activities may play important roles in Cd-stressed Fuhong 991 and ZM 412 and that the leaf and root cell membranes of Fuhong 991 have a greater stability than those of ZM 412. For pollution monitoring purposes, the GR activity in the roots and leaves may serve as a biomarker of Cd for Fuhong 991, whereas lipid peroxidation may serve as biomarker for ZM 412.

Isolation and analysis of a TIR-specific promoter from poplar

A 5＇ flanking region of the well-conserved Toll/interleukin-1 receptor domain （TIR）-encoding sequence was isolated from the genomic DNA ofMelampsora magnusiana Wagner resistant clones of hybrid triploid poplars [（Populus tomentosa × P. bolleana） × P. tomentosa]. Sequencing results and alignment analysis show that the obtained TIR-specific promoter （named as PtTIRp01） was 1,732 bp in length; moreover 3＇ region of the PtTIRp01 contains a responds to the 5＇ composition of TIR-NBS type gene PtDRG02, of 747 bp long 5＇ region of TIR-NBS type gene PtDRG02 and its 398 bp complete TIR-encoding sequence, which significantly corindicating that the obtained TIR-specific promoter region consists upstream region of promoter （985 bp）. It was found that the 5＇ region of TIR-NBS type gene PtDRG02 was characterized in the downstream region of the transcriptional start, named as 5＇-untranslated region （5＇ UTR）, consisting of one 93 bp 5＇-untranslation exon, one 213 bp intron and one 441 bp TIR-encoding open reading frame （ORF）. In addition, several putative cis-acting motifs were present in the obtained TIR-specific promoter of PtDRG02, including one TATA box, one GC-rich, one AT-rich, one P-box, one 3-AF1 binding site, two CAAT boxes, two GT-1 motifs, three typical W-boxes, four I-boxes, and one multi-cis-acting fragment （MCF）. The latter contains five types of regulatory elements （E4, G-box, ABRE motif, box 1 and HVA 1 s）, most of which were homologous to the cis-acting regulatory elements involved in the activation of defense genes in plants. Thus, it can be suggested that TIR-specific promoter might be a pathogen-inducible promoter and be necessary for the inducible expression of defense-related genes. Key words Toll/interleukin- 1 receptor domain, promoter, Cis-acting element, poplar

Mapping of Mutant Gene prbs Controlling Poly-Row-and-Branched Spike in Barley (Hordeum vulgare L.)

A row-type mutant of barley named poly-row-and-branched spike （prbs） was previously obtained from a two-rowed cultivar Pudamai-2 after treated by inflorescence soaking in maize total DNA solution. The mutant produces branched spikes with irregular multiple rows. Genetic analysis indicated that the mutant phenotype was caused by a recessive gene prbs, and the PRBS locus had a recessive epistatic effect on an independent locus （denoted as Vrsx） conferring the variation of two-rowed spike vs. six-rowed spike. This study aimed to map PRBS as well as Vrsx using simple sequence repeats （SSR） markers. We developed an F2 population from a cross between the prbs mutant and a six-rowed cultivar Putianwudu for the gene mapping. As the two target loci interacted to result in a segregation ratio of two-rowed type：six-rowed type：prbs=9：3：4 in the population, we adopted a special strategy to map the two loci. PRBS was mapped between SSR markers HvLTPPB and Bmag0508A on the short arm of chromosome 3H, with distances of 24.7 and 14.3 cM to the two markers, respectively. Vrsx was mapped between SSR markers Bmag0125 and Bmag0378 on chromosome 2H, with distances of 6.9 and 15.3 cM to the two markers, respectively. This suggests that Vrsx should be the known locus Vrs1, which predominantly controls row-type variation in barley cultivars, and PRBS is a new locus related to the row type of spikes in barley.

Optimal management of nitrogen fertilizer in the main rice crop and its carrying-over effect on ratoon rice under mechanized cultivation in Southeast China

This study attempted to clarify the carrying-over effect of different nitrogen treatments applied to the main crop on the crop population growth and yield formation of ratoon rice under mechanized cultivation in Southeast China.Based on the constant total nitrogen application amounts(225.00 kg ha;)in the main crop,an experiment with different ratios of basal and topdressing nitrogen fertilizer(the ratio of basal fertilizer:primary tillering fertilizer:secondary tillering fertilizer:booting fertilizer at 3:1:2:4(N1),3:2:1:4(N2),3:3:0:4(N3),and 4:3:0:3(N4),respectively,and a control without nitrogen treatment(N0))was set up across two consecutive years in field using hybrid rice variety Yongyou 1540 as the test materials.The results showed that the total tiller number and effective tillering percentage increased in the main crop under the N1 treatment,more nitrogen fertilizer applied in late growth stage of the main crop,and its effective tillering percentage of the main crop was the highest at up to 70.18%,which was 9.15%higher than that of conventional fertilization treatment(N4),more nitrogen fertilizer applied in early growth stage of the main crop.The same tendency was observed in leaf area index(LAI)value of the main crop and its subsequent ratoon rice,which were 16.52 and 29.87%higher,respectively,in the N1 treatment than that in the N4 treatment at the full heading stage.The same was true in the case of the transport rates of stem and sheath dry mater and the canopy light interception rates in both the main and its ratoon crops.The transport rate of stem and sheath in main crop rice under N1 treatment increased by 50.57%compared with N4 treatment.The canopy light interception rate of N1 treatment increased by 5.07%compared with N4 treatment at the full heading stage of the ratoon crop.Therefore,the total actual yield was the highest in the main and its ratoon crops under N1 treatment,averaging 17351.23 kg ha;in two-year trials,which was 23.00%higher than that in the conventional fertilization treatment(N4).The results showed that appropriate nitrogen treatment was able to produce a good crop stand in the main crop,which was essential for producing a good ratoon crop population and high yield especially under mechanized cultivation with low stubble height of the main crop.The study suggested that shifting the proper nitrogen application amounts to the late growth stage of the main crop,such as N1 treatment,not only had a higher productive effect on ensuring the yield of the main crop,but also had a positive effect on the axillary bud sprouts from the stubbles for ratoon rice,resulting in an increased percentage of productive panicles and achieving the goal of one planting with two good harvests under the conditions of our study.

Identification and characterization of CBF/DREB1-related genes in Populus hopeiensis

The dehydration-responsive element-binding factor （DREB） is a plant-specific family of transcription factors and plays an important role in plant response and adaptation to abiotic stress. In the present work, two highly similar CBF/DREB l-like genes, designated as PhCBF4a and PhCBF4b, were identified from Populus hopeiensis. These two genes contain all conserved domains known to exist in other CBF/DREB1 genes. In the AP2 domain, there is only one different amino acid residue between PhCBF4a and PhCBF4b, alanine or valine, a nonpolar amino acid, suggesting that PhCBF4a and PhCBF4b may have similar DNA binding ability. Their expression is induced by water-loss treatment and their expression patterns are similar. Moreover, with a genomic DNA as template, the presence of the same bands in PCR products as those in expression pattern analysis indicated that PhCBF4a and PhCBF4b exist in the genome ofP. hopeiensis. Their detailed functions are discussed and will need further study.

A Genetic Linkage Map of Kenaf (Hibiscus cannabinus L.) Based on SRAP, ISSR and RAPD Markers

Kenaf （Hibiscus cannabinus L.） is one of the most economically important crops for non-wood fiber production. The objective of this study was to establish a genetic linkage map of kenaf with higher density of molecular markers. A semi- wild variety Ga42 and a cultivar Alain kenaf were used as parents to construct an F2 population consisting of 155 plants. The genetic linkage map comprising 134 marker loci was constructed, including 65 sequence-related amplified polymorphism （SRAP）, 56 inter-simple sequence repeat （ISSR）, and 13 randomly amplified polymorphic DNA （RAPD） markers. This map spans 2 108.9 cM and contains 20 linkage groups with an average marker density of 15.7 cM between the adjacent markers.

RNA interference and its application in plants

RNA interference （RNAi）, a process that inhibits gene expression by the double-stranded RNA （dsRNA）, causes the degradation of target messenger RNA molecules. RNAi exists in almost all organisms. We review the recent history of RNAi studies, RNAi molecular mechanisms, characteristics and RNAi applications in higher plants. At the same time, the prospect of RNAi applications in functional genomics and genetic improvement of higher plants and possible future problems and possibilities are also discussed.

DNA recovery from agarose gels with a simple centrifuge-driven sephadex filtration

Conventional methods of DNA recovery from agarose gel generally require expensive equipment, extended elution times, or considerable handling of the sample after elution. We developed a simple protocol for a quick and effective recovery of DNA from agarose gels with good yield and quality. Using a Sephadex resin filled spin column, DNA fragments of 500 bp to 6 kb in an agarose gel slice were easily recovered by a 2 min centrifugation. The recovery efficiencies were over 40% -50% and the eluted DNA can be used directly for downstream application, such as polymerase chain reactions （PCR） and restriction enzyme digestion. This method could also be used to recover large DNA fragment （48 kb） without degradation. The use of Sephadex helps to remove small molecular impurities from agarose and it also reduces the chance of clogging the column filter caused by direct contact with agarose.

Genetic structure of needle morphological and anatomical traits of Pinus yunnanensis

Pinus yunnanensis Franch. is an particular conifer tree species in Yunnan-Guizhou plateau in south- west China. The morphological and anatomical traits of needles are important to evaluate geographic variation and population dynamics of conifer species. Seedlings from seven populations of P. yunnanensis were analyzed, look- ing at 22 morphological and anatomical needle traits. The results showed that variations among and within popula- tions were significantly different for all traits and the variance components within populations were generally higher than that among populations in the most tested needle traits. The proportions of three-needle fascicle were significantly different among populations. The traits related to needle size in both morphology and anatomy were positive with latitude and negative with annual temperature and precipitation. Ratio indices, including mesophyllarea/vascular bundle area, mesophyll area/resin canals area, vascular bundle area/resin canals area and mesophyU area/（resin canals area and vascular bundle area）, were negatively correlated with elevation and positively corre- lated with the annual mean temperature, showing some fitness feature for the populations. Needle traits were more significantly correlated with longitude than with other four environmental factors. Needle length was significantly correlated with almost all environmental factors. First four principal components accounted for 81.596 % of the variation with eigenvalues 〉1; the differences among populations were mainly dependent on needle width, stomatal density, section areas of vascular bundle, total resin canals, and mesophyll, as well as area ratio traits. Seven populations were divided into three categories by Euclidean distance. Variations in needle traits among the populations have shown systematic microevolution in terms of geographic impact on P. yunnanensis. This study would provide empirical data to characterize adaptation and genetic variation of P. yunnanensis, which would be helpful for management of genetic resources and reason- able utilization of them in future.

Effects of different supplements on tetraploid black locust (Robinia pseudoacacia L.) silage

Preparation of silage is a common method to preserve green forage. It plays an important role in improving forage utilization, solving the problem of forage provision and meeting the nutritional needs of livestock in winter and spring. The effects of various supplements on tetraploid black locust （Robinia pseudoacacia L.） silage were studied by analyzing its color, odor, texture, pH value, the composition and amount of organic acids, the ammonia-N/total-N ratio, crude protein, neutral detergent fibers and acid detergent fibers. Our results show that the silage quality of wilted tetraploid R. pseudoacacia preserved alone is acceptable, while adding brown sugar and Yishengkang to tetraploid R. pseudoacacia foliage improved its quality. The treatments consisting of the tet- raploid R. pseudoacacia mixed with 33% and 50% corn stalk achieved the best silage quality in terms ofpH value （p 〈 0.01）, amount of lactic acid （p 〈 0.01）, the ammonia-N/total-N ratio （p 〈 0.01） and the fermentation quality. These treatments also resulted in a high content of crude protein and lower amounts of acid detergent fibers. The treatment consisting of the tetraploid R. pseudoacacia mixed with 20% corn stalk was also good in the quality of its fermentation.

The diverse roles of cytokinins in regulating leaf development

Leaves provide energy for plants,and consequently for animals,through photosynthesis.Despite their important functions,plant leaf developmental processes and their underlying mechanisms have not been well characterized.Here,we provide a holistic description of leaf developmental processes that is centered on cytokinins and their signaling functions.Cytokinins maintain the growth potential(pluripotency)of shoot apical meristems,which provide stem cells for the generation of leaf primordia during the initial stage of leaf formation;cytokinins and auxins,as well as their interaction,determine the phyllotaxis pattern.The activities of cytokinins in various regions of the leaf,especially at the margins,collectively determine the final leaf morphology(e.g.,simple or compound).The area of a leaf is generally determined by the number and size of the cells in the leaf.Cytokinins promote cell division and increase cell expansion during the proliferation and expansion stages of leaf cell development,respectively.During leaf senescence,cytokinins reduce sugar accumulation,increase chlorophyll synthesis,and prolong the leaf photosynthetic period.We also briefly describe the roles of other hormones,including auxin and ethylene,during the whole leaf developmental process.In this study,we review the regulatory roles of cytokinins in various leaf developmental stages,with a focus on cytokinin metabolism and signal transduction processes,in order to shed light on the molecular mechanisms underlying leaf development.

Characteristics of nucleotides and amino acids in cDNA sequence of xyloglucan endotransglycosylase cloned from Anthocephalus chinensis

Xyloglucan endotransglycosylase is an identified relaxation factor with functions of easing and extending of plant cell walls.Its activities are directly related to plant growth and elongation of organisms.Anthocephalus chinensis is a tall and fast growing evergreen tree species.We cloned the full cDNA sequence of AcEXT genes which is abundantly expressed in the cambium of A.chinensis.The sequence analysis of nucleotides and amino acids revealed the presence of a 1396 bp full cDNA sequence,including a 960 bp complete open reading frame（ORF） encoding a 320 amino acid protein.The deduced amino acid sequence of AcXET was homologous to the other known XET proteins and contained the conserved EIDFE catalytic site which was specific to all the XETs.Our data should serve as a foundation for further insight into AcXET gene molecular mechanisms during wood formation and cell wall engineering of woody plants.

Effect of NaCl on growth and ion relations in two salt-tolerant strains of Paxillus involutus

The effect of NaCl on growth, biomass and ion relations of two salt-tolerant isolates of Paxillus involutus, MAJ and NAU were investigated. The two Paxillus strains were exposed to the following concentrations of NaCl： 0, 100, 200 and 500 mmol·L^-1. Growth of MAJ and NAU was enhanced by 100 mmol·L^-1 NaCl but severely inhibited at the concentration of 500 mmol·L^-1. NAU exhibited a greater capacity to exclude Na^＋ and Cl^- under all salinity levels, whereas the salt-includer MAJ had a higher capacity in nutrient uptake under salt stress. The ratios Na^＋/K^＋, Na^＋/Ca^2＋ and Na^＋/Mg^2＋ in NaCl-treated MAJ and NAU did not increase at levels of 100 and 200 mmol·L^-1 NaCl but markedly increased at 500 mmol·L^-1. This suggests that the two strains, especially MAJ, enhanced nutrient uptake corresponding to the increased Na^＋ influx at moderate salinity. We conclude that both MAJ and NAU are able to tolerate 500 mmol·L^-1 NaCl but there are species-specific differences in retaining ionic homeostasis in the two Paxillus strains. NAU is a salt-excluder, MAJ is a salt-includer but retains a high capacity in nutrient selectivity under saline conditions. Their definite mechanisms to enhance salt tolerance of mycorrhizal hosts need further study.

Pollen development and multi-nucleate microspores of Populus bolleana Lauche

Populus bolleana is a variety of P. alba, commonly used in poplar breeding programs in China. Developmental biology that involves staminate flowers, microsporogenesis and microgametogenesis ofP. bolleana is essential for Populus improvement in cross breeding for better characteristics in sexual reproduction. Flower morphology and pollen development were described and illustrated using anatomical, sectioning and stain-clearing techniques. The results show that microsporocytes undergo a regular meiotic process, but some multi-nucleate microspores occur at the microspore stage. It takes five days for microsporocytes to develop to mature pollen by forcing flower branches under greenhouse conditions. Additionally, an important relationship was found between stages of meiosis and anther colors. Microspore tetrads formed when the anther color turned yellow, whereas, when the pollen matured, the anther was red and the tapetum degenerated completely. When mature pollen grains are formed, flower buds develop into male catkins. In the end, filament elongated and pollen grains were released from dehisced anthers.

Comparative microsporogenesis and flower development in Eucalyptus urophylla×E.grandis

Microsporogenesis and flower development in Eucalyptus urophylla × E. grandis were examined using chromosome tableting to provide a method to predict the meiotic stages in this species. Although microsporogenesis was normal, cytokinesis during meiosis of pollen mother cells occurred simultaneously, with strong asynchronism observed in the two different lengths of stamens in a flower bud. In a single flower, the developmental period of microsporogenesis in anthers on the longer stamens was always ahead of those on the shorter stamens. Flower development was also asynchronous at different locations on a branch. Flower buds on the upper side of the branch were larger in diameter than those on the lower side. In addition, a correlation was observed between microsporogenesis development and flower bud diameter growth. The pachy- tene stage was first observed when the diameter of the flower buds increased to 3.0 mm, and the majority of the meiotic stages were observed when bud diameters ranged from 3.5 to 5.0 mm. This study showed that the developmental stages of microsporogenesis in Eucalyptus urophylla × E. grandis could be distinguished readily, which may be applicable to future breeding studies.