Construction and analysis of a plant transformation binary vector pBDGG harboring a bi-directional promoter fusing dual visible reporter genes

The constitutive promoter of cauliflower mosaic virus 35S （CaMV 35S） is a polar unidirectional promoter and is widely used in plant genetic engineering. In the present study, the unidirectional CaMV 35S promoter has been modified to a bi-directional promoter by fusing its minimal promoter element to the 5＇ end of CaMV 35S promoter in the opposite orientation. To qualitatively and quantitatively estimate its bi-directional transcriptional function and activity, two visible reporter genes, gusA （13-glucuronidase, GUS） and gfp （green fluorescent protein, GFP）, were fused to the two ends of the promoter in bi-orientations ending with NOS terminator sequences, respectively. Stable expression of gusA and gfp genes in transgenic tobacco （Nicotiana tabacum L.） was visulized by histochemically staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants. The expression of two reporter genes showed that the constructed bi-directional promoter did have the bi-directional transcriptional function in both expected orientations. The quantitative estimation of GUS and GFP were determined on a HITACHI F1000 Fluorescence Spectrophotometer with various wavelengths of excitation and emission. The GUS activity varied from g to 250 pmol 4-MU/min/mg protein and the GFP content varied from 0.9 to 1.8 μg/ mg protein in various lines of transgenic tobacco plants. Higher GUS activity generally coupled with lower GFP content, and vice versa.

Adaptive strategy of Nitraria sibirica to transient salt,alkali and osmotic stresses via the alteration of Na+/K+fluxes around root tips

Nitraria sibirica Pall.is an important shrub with a strong salt-alkali tolerance,but the mechanism underlying this tolerance remains obscure.In this study,N.sibirica,with salt-sensitive Vigna radiata(Linn.)Wilczek as the control,was subjected to transient salt stress(100 mM NaCl),alkali stress(50 mM Na_(2)CO_(3)),and osmotic stress(175 mM mannitol).The ionic fluxes of Na^(+)and K^(+)in the root apical region were measured.Results show that,under salt and alkali stress,N.sibirica roots exhibited higher capacities to limit Na+influx and reduce K+efflux,thereby resulting in lower Na^(+)/K^(+)ratios compared with V.radiata roots.Alkali stress induced stronger Na^(+)influx and K+efflux in the root salt stress treatment;Na^(+)influx was mainly observed in the root cap,while K^(+)efflux was mainly observed in the elongation zone.While under osmotic stress,N.sibirica roots showed stronger Na+efflux and weaker K+efflux than V.radiata roots.Na+efflux was mainly observed in the root elongation zone,while K+efflux was in the root cap.These results reveal the ionic strategy of N.sibirica in response to transient salt,alkali,and osmotic stresses through the regulation of Na+/K+flux homeostasis.

Age–age correlations and early selection for growth traits in 40 half-sib families of Larix principis-rupprechtii

Larix principis-rupprechtii Mayr is a dominant species in coniferous forests of North China.However,early selection of L.principis-rupprechtii for growth traits is poorly characterised.To explore the optimal selection age for this species,heights(HT)and diameters at breast(DBH)of 40 half-sib families were measured at ages 3,12,22,and 28 years in a progeny test population established in the town of Kangjiahui,Shanxi Province.Age trends in heritability,age–age genetic correlations,and early selection efficiency for height and DBH were analysed.The individual heritability of these growth traits varied over time,and maximized at different ages(0.55 at age 12 for HT and 0.48 at age 28 for DBH).The age–age genetic correlations were always positive,and the majority were high(0.790–0.953)between the juvenile and mature ages for HT and DBH.For the same pairs of measurements,HT demonstrated higher age–age genetic correlations than DBH,and both age–age genetic correlation data sets were described well by the linear relationship with the logarithm of the age ratio(r2[0.90).The regression slope for DBH was lower than that for HT.Based on the early selection efficiency estimates,the optimal selection age could be as early as age 6 for DBH and 8–9 years for HT.The results of this study provide information that can be used to assist early selection practices in L.principis-rupprechtii improvement programs in Shanxi Province.

Morphologic characters and element content during development of Pinus tabuliformis seeds

An embryo classification system for Pinus tabuliformis Carr. was established by time-tracing sam- pling and observation of zygotic embryos. The zygotic embryos were divided into nine stages. Key elements of the zygotic embryo and female gametophyte （FG） tissue of P. tabuliformis were analyzed, using inductively coupled plasma-emission spectroscopy. Several elements--includ- ing aluminum, iron, sodium, and copper--are found in both embryo and FG tissue. Boron, phosphorus, magnesium,zinc, and calcium are also required for zygotic embryo development and therefore accumulated. Manganese is selectively excluded from the embryo. The zygotic embryo development needs a low-sodium and high-potassium nutrition proportion. The results of elemental analysis from zygotic embryos and FGs can provide the mineral targets for optimizing the formulation of culture medium for somatic embryogenesis.

Identification and Characterization of the Populus AREB/ABF Subfamily

Abscisic acid （ABA） is a major plant hormone that plays an important role in responses to abiotic stresses. The ABA-responsive element binding protein/ABRE-binding factor （AREB/ABF） gene subfamily contains crucial transcription factors in the ABA-mediated signaling pathway. In this study, a total of 14 putative AREB/ABF members were identified in the Populus trichocarpa Torr. ＆ Gray. genome using five AREB/ABF amino acid sequences from Arabidopsis thaliana L. as probes. The 14 putative Populus subfamily members showed high protein similarities, especially in the basic leucine zipper （bZIP） domain region. A neighbor-joining analysis combined with gene structure data revealed homology among the 14 genes. The expression patterns of the Populus AREB/ABF subfamily suggested that the most abundant transcripts of 11 genes occurred in leaf tissues, while two genes were most transcribed in root tissues. Significantly, eight Populus AREB/ABF gene members were upregulated after treatment with 100 t^M exogenous ABA, while the other six members were downregulated. We identified the expression profiles of the subfamily members in Populus tissues and elucidated different response patterns of Populus AREB/ABF members to ABA stress. This study provided insight into the roles of Populus AREB/ABF homologues in plant response to abiotic stresses.

Differences in leaf cuticular wax induced by whole-genome duplication in autotetraploid sour jujube

Drought-resistant plants exhibit strong water retention capability.In this regard,the autotetraploid sour jujube leaves exhibit better water retention than diploid leaves.Morphological comparisons and physiological comparisons of diploid and autotetraploid leaves showed that the autotetraploid leaves had thicker leaf cuticles and more leaf wax accumulation than the diploid leaves,which could reduce cuticle permeability and improve the drought tolerance of leaves.In this study,the cuticular wax crystalloids on the adaxial and abaxial sides of young and mature jujube leaves were observed in the two ploidy types,and unique cuticular wax crystalloids covering a large area of the cuticle on autotetraploid sour jujube leaves may provide an advantage in reducing leaf non-stomata transpiration and improving plant drought tolerance.Based on the transcriptome,115 differentially expressed genes between diploids and autotetraploids were further analyzed and found to be involved in the accumulation of cuticular wax components,including terpenoids,fatty acids,and lipids,as well as ABC transporter and wax biosynthetic process.Finally,14 genes differentially expressed between glossy autotetraploid leaves and nonglossy diploid leaves,such as LOC107414787,LOC107411574 and LOC107413721,were screened as candidate genes by qRT-PCR analysis.This findings provided insights into how polyploidization improved drought tolerance.

Function identification of miR159a,a positive regulator during poplar resistance to drought stress

Drought seriously affects the growth and development of plants.MiR159 is a highly conserved and abundant microRNA family that plays a crucial role in plant growth and stress responses.However,studies of its function in woody plants are still lacking.Here,the expression of miR159a was significantly upregulated after drought treatment in poplar,and the overexpression of miR159a(OX159a)significantly reduced the open area of the stomata and improved water-use efficiency in poplar.After drought treatment,OX159a lines had better scavenging ability of reactive oxygen species and damage of the membrane system was less than that in wild-type lines.MYB was the target gene of miR159a,as verified by psRNATarget prediction,RT-qPCR,degradome sequencing,and 5′rapid amplification of cDNA ends(5′RACE).Additionally,miR159a-short tandem target mimic suppression(STTM)poplar lines showed increased sensitivity to drought stress.Transcriptomic analysis comparing OX159a lines with wild-type lines revealed upregulation of a series of genes related to response to water deprivation and metabolite synthesis.Moreover,drought-responsive miR172d and miR398 were significantly upregulated and downregulated respectively in OX159a lines.This investigation demonstrated that miR159a played a key role in the tolerance of poplar to drought by reducing stomata open area,increasing the number and total area of xylem vessels,and enhancing water-use efficiency,and provided new insights into the role of plant miR159a and crucial candidate genes for the molecular breeding of trees with tolerance to drought stress.

MicroRNAs as potent regulators in nitrogen and phosphorus signaling transduction and their applications

Nitrogen(N)and phosphorus(Pi)are essential macronutrients that affect plant growth and development by influencing the molecular,metabolic,biochemical,and physiological responses at the local and whole levels in plants.N and Pi stresses suppress the physiological activities of plants,resulting in agricultural productivity losses and severely threatening food security.Accordingly,plants have elaborated diverse strategies to cope with N and Pi stresses through maintaining N and Pi homeostasis.MicroRNAs(miRNAs)as potent regulators fine-tune N and Pi signaling transduction that are distinct and indivisible from each other.Specific signals,such as noncoding RNAs(ncRNAs),interact with miRNAs and add to the complexity of regulation.Elucidation of the mechanisms by which miRNAs regulate N and Pi signaling transduction aids in the breeding of plants with strong tolerance to N and Pi stresses and high N and Pi use efficiency by fine-tuning MIR genes or miRNAs.However,to date,there has been no detailed and systematic introduction and comparison of the functions of miRNAs in N and Pi signaling transduction from the perspective of miRNAs and their applications.Here,we summarized and discussed current advances in the involvement of miRNAs in N and Pi signaling transduction and highlighted that fine-tuning the MIR genes or miRNAs involved in maintaining N and Pi homeostasis might provide valuable sights for sustainable agriculture.