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Silique walls play pivotal roles in contributing photoassimilates and nutrients to fuel seed growth. However, the interaction between seeds and silique walls impacting oil biosynthesis is not clear during silique development. Changes in sugar, fatty acid and gene expression during Brassica napus silique development of L192 with high oil content and A260 with low oil content were investigated to identify key factors affecting difference of their seed oil content. During the silique development, silique walls contained more hexose and less sucrose than seeds, and glucose and fructose contents in seeds and silique walls of L192 were higher than that of A260 at 15 DAF, and sucrose content in the silique walls of L192 were lower than that of A260 at three time points. Genes related to fatty acid biosynthesis were activated over time, and differences on fatty acid content between the two genotypes occurred after 25 DAF. Genes related to photosynthesis expressed more highly in silique walls than in contemporaneous seeds, and were inhibited over time. Gene set enrichment analysis suggested photosynthesis were activated in L192 at 25 and 35 DAF in silique walls and at both 15 and 35 DAF in the seed. Expressions of sugar transporter genes in L192 was higher than that in A260, especially at 35 DAF. Expressions of genes related to fatty acid biosynthesis, such as BCCP2s, bZIP67 and LEC1s were higher in L192 than in A260, especially at 35 DAF. Meanwhile, genes related to oil body proteins were expressed at much lower levels in L192 than in A260. According to the WGCNA results, hub modules, such as ME.turquoise relative to photosynthesis, ME.green relative to embryo development and ME.yellow relative to lipid biosynthesis, were identified and synergistically regulated seed development and oil accumulation. Our results are helpful for understanding the mechanism of oil accumulation of seeds in oilseed rape for seed oil content improvement.

Polyploidy is a common mode of evolution in flowering plants. Both the natural tetraploid Thinopyrum elongatum and the diploid one from the same population show a diploid-like pairing in meiosis. However, debate on the chromosome composition and origin of the tetraploid Th. elongatum is ongoing. In the present study, we obtained the induced tetraploid Th. elongatum and found that the induced and natural tetraploids are morphologically close, except for slower development and lower seed setting. Using probes developed from single chromosome microdissection and a Fosmid library, obvious differentiations were discovered between two chromosome sets (E

Triacylglycerol (TAG) is the most important storage lipid for oil plant seeds. Diacylglycerol acyltransferases (DGATs) are a key group of rate-limiting enzymes in the pathway of TAG biosynthesis. In plants, there are three types of DGATs, namely, DGAT1, DGAT2 and DGAT3. Brassica napus, an allotetraploid plant, is one of the most important oil plants in the world. Previous studies of Brassica napus DGATs (BnaDGATs) have mainly focused on BnaDGAT1s. In this study, four DGAT1s, four DGAT2s and two DGAT3s were identified and cloned from B. napus ZS11. The analyses of sequence identity, chromosomal location and collinearity, phylogenetic tree, exon/intron gene structures, conserved domains and motifs, and transmembrane domain (TMD) revealed that BnaDGAT1, BnaDGAT2 and BnaDGAT3 were derived from three different ancestors and shared little similarity in gene and protein structures. Overexpressing BnaDGATs showed that only four BnaDGAT1s can restore TAG synthesis in yeast H1246 and promote the accumulation of fatty acids in yeast H1246 and INVSc1, suggesting that the three BnaDGAT subfamilies had greater differentiation in function. Transcriptional analysis showed that the expression levels of BnaDGAT1s, BnaDGAT2s and BnaDGAT3s were different during plant development and under different stresses. In addition, analysis of fatty acid contents in roots, stems and leaves under abiotic stresses revealed that P starvation can promote the accumulation of fatty acids, but no obvious relationship was shown between the accumulation of fatty acids with the expression of BnaDGATs under P starvation. This study provides an extensive evaluation of BnaDGATs and a useful foundation for dissecting the functions of BnaDGATs in biochemical and physiological processes.

Plant artificial minichromosomes are the next-generation technology for plant genetic engineering and represent an independent platform for expressing foreign genes and the tools for studying the structure and function of chromosomes. Minichromosomes have been successfully produced by telomere-mediated chromosome truncation in several plants. However, previous studies have primarily focused on the construction and rough characterization of minichromosomes, while the development of stably inherited minichromosomes and their precise characterization and tracking over different generations have rarely been demonstrated. In this study, a 0.35-kb direct repeat of the Arabidopsis telomeric sequence was transformed into Brassica napus to produce artificial minichromosomes, which were analyzed by multifluorescence in situ hybridization (multi-FISH), Southern hybridization, and primer extension telomere rapid amplification (PETRA). The stably inherited minichromosomes C2 and C4 were developed by crossing transgenic plants with wild-type plants and then selfing the hybrids. Notably, two truncation sites on chromosomes C2 and C4, respectively, were identified by resequencing; thus, the artificial minichromosomes were tracked over different generations with insertion site-specific PCR. This study provided two stably inherited minichromosomes in oilseed rape and describes approaches to precisely characterize the truncation position and track the minichromosomes in offspring through multi-FISH, genome resequencing, and insertion site-specific PCR.

Proline-rich protein (PRP) is a plant cell wall associated protein. Its distinct patterns of regulation and localization studied in a number of plants indicate that it may play important roles in growth and development. However, the mechanism of how these genes control secondary cell wall development in tree species is largely unknown. Here, we report that a Populus deltoides (Marsh.) proline-rich protein gene PdPRP was preferentially expressed in immature/mature phloem and immature xylem in P. deltoides. PdPRP overexpression increased poplar plant height and diameter as well as the radial width of the phloem and xylem regions, facilitated secondary wall deposition, and induced expression of genes related to microfibril angle (MFA) and secondary wall biosynthesis. Downregulation of PdPRP retarded poplar growth, decreased the radial width of the secondary phloem and secondary xylem regions, reduced secondary wall thickening in fibers and vessels, and decreased the expression of genes related to MFA and secondary wall biosynthesis. These results suggest that PdPRP might positively regulate secondary cell wall formation by promoting secondary wall thickening and expansion in poplar. PdPRP-overexpressing poplar had a lower MFA, indicating that PdPRP may be useful for improving wood stiffness and properties in plants. Together, our results demonstrate that PdPRP is a proline-rich protein associated with cell wall development, playing a critical role in regulating secondary cell wall formation in poplar.

Different types of P genome sequences and markers were developed, which could be used to analyze the evolution of P genome in Triticeae and identify precisely wheat- A. cristatum introgression lines. P genome of Agropyron Gaertn. plays an important role in Triticeae and could provide many desirable genes conferring high yield, disease resistance, and stress tolerance for wheat genetic improvement. Therefore, it is significant to develop specific sequences and functional markers of P genome. In this study, 126 sequences were isolated from the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) products of microdissected chromosome 6PS. Forty-eight sequences were identified as P genome-specific sequences by dot-blot hybridization and DNA sequences analysis. Among these sequences, 22 displayed the characteristics of retrotransposons, nine and one displayed the characteristics of DNA transposons and tandem repetitive sequence, respectively. Fourteen of 48 sequences were determined to distribute on different regions of P genome chromosomes by fluorescence in situ hybridization, and the distributing regions were as following: all over P genome chromosomes, centromeres, pericentromeric regions, distal regions, and terminal regions. We compared the P genome sequences with other genome sequences of Triticeae and found that the similar sequences of the P genome sequences were widespread in Triticeae, but differentiation occurred to various extents. Additionally, thirty-four molecular markers were developed from the P genome sequences, which could be used for analyzing the evolutionary relationship among 16 genomes of 18 species in Triticeae and identifying P genome chromatin in wheat-A. cristatum introgression lines. These results will not only facilitate the study of structure and evolution of P genome chromosomes, but also provide a rapid detecting tool for effective utilization of desirable genes of P genome in wheat improvement.

∆(8)-sphingolipid desaturase catalyzes the C8 desaturation of a long chain base, which is the characteristic structure of various complex sphingolipids. The genes of 20 ∆(8)-sphingolipid desaturases from 12 plants were identified and functionally detected by using Saccharomyces cerevisiae system to elucidate the relationship between the biochemical function and evolution of this enzyme. Results showed that the 20 genes all can encode a functional ∆(8)-sphingolipid desaturase, which catalyzes different ratios of two products, namely, 8(Z) and 8(E)-C18-phytosphingenine. The coded enzymes could be divided into two groups on the basis of biochemical functions: ∆(8)-sphingolipid desaturase with a preference for an E-isomer product and ∆(8)-sphingolipid desaturase with a preference for a Z-isomer product. The conversion rate of the latter was generally lower than that of the former. Phylogenetic analysis revealed that the 20 desaturases could also be clustered into two groups, and this grouping is consistent with that of the biochemical functions. Thus, the biochemical function of ∆(8)-sphingolipid desaturase is correlated with its evolution. The two groups of ∆(8)-sphingolipid desaturases could arise from distinct ancestors in higher plants. However, they might have initially evolved from ∆(8)-sphingolipid desaturases in lower organisms, such as yeasts, which can produce E-isomer products only. Furthermore, almost all of the transgenic yeasts harboring ∆(8)-sphingolipid desaturase genes exhibit an improvement in aluminum tolerance. Our study provided new insights into the biochemical function and evolution of ∆(8)-sphingolipid desaturases in plants.

Transposable elements (TEs) in plant genomes exhibit a great variety of structure, sequence content and copy number, making them important drivers for species diversity and genome evolution. Even though a genome-wide statistic summary of TEs in rye has been obtained using high-throughput DNA sequencing technology, the accurate diversity of TEs in rye, as well as their chromosomal distribution and evolution, remains elusive due to the repetitive sequence assembling problems and the high dynamic and nested nature of TEs. In this study, using genomic plasmid library construction combined with dot-blot hybridization and fluorescence in situ hybridization (FISH) analysis, we successfully isolated 70 unique FISH-positive TE-related sequences including 47 rye genome specific ones: 30 showed homology or partial homology with previously FISH characterized sequences and 40 have not been characterized. Among the 70 sequences, 48 sequences carried Ty3/gypsy-derived segments, 7 sequences carried Ty1/copia-derived segments and 15 sequences carried segments homologous with multiple TE families. 26 TE lineages were found in the 70 sequences, and among these lineages, Wilma was found in sequences dispersed in all chromosome regions except telomeric positions; Abiba was found in sequences predominantly located at pericentromeric and centromeric positions; Wis, Carmilla, and Inga were found in sequences displaying signals dispersed from distal regions toward pericentromeric positions; except DNA transposon lineages, all the other lineages were found in sequences displaying signals dispersed from proximal regions toward distal regions. A high percentage (21.4%) of chimeric sequences were identified in this study and their high abundance in rye genome suggested that new TEs might form through recombination and nested transposition. Our results also gave proofs that diverse TE lineages were arranged at centromeric and pericentromeric positions in rye, and lineages like Abiba might play a role in their structural organization and function. All these results might help in understanding the diversity and evolution of TEs in rye, as well as their driving forces in rye genome organization and evolution.

Male sterility in plants has been strongly linked to mitochondrial dysfunction. Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. Therefore, it is important to better understand the relationship between mitochondria and CHA-induced male sterility in wheat. This study reports on the impairment of mitochondrial function duo to CHA-SQ-1, which occurs by decreasing cytochrome oxidase and adenosine triphosphate synthase protein levels and theirs activities, respiratory rate, and in turn results in the inhibition of the mitochondrial electron transport chain (ETC), excessive production of reactive oxygen species (ROS) and disruption of the alternative oxidase pathway. Subsequently, excessive ROS combined with MnSOD defects results in damage to the mitochondrial membrane, followed by ROS release into the cytoplasm. The microspores underwent severe oxidative stress during pollen development. Furthermore, chronic oxidative stress, together with the overexpression of type II metacaspase, triggered premature tapetal apoptosis, which resulted in pollen abortion. Accordingly, we propose a metabolic pathway for mitochondrial-mediated male sterility in wheat, which provides information on the molecular events underlying CHA-SQ-1-induced abortion of anthers and may serve as an additional guide to the practical application of hybrid breeding.

Δ8-sphingolipid desaturase and Δ6-fatty acid desaturase share high protein sequence identity. Thus, it has been hypothesized that Δ6-fatty acid desaturase is derived from Δ8-sphingolipid desaturase; however, there is no direct proof. The substrate recognition regions of Δ6-fatty acid desaturase and Δ8-sphingolipid desaturase, which aid in understanding the evolution of these two enzymes, have not been reported. A blackcurrant Δ6-fatty acid desaturase and a Δ8-sphingolipid desaturase gene, RnD6C and RnD8A, respectively, share more than 80 % identity in their coding protein sequences. In this study, a set of fusion genes of RnD6C and RnD8A were constructed and expressed in yeast. The Δ6- and Δ8-desaturase activities of the fusion proteins were characterized. Our results indicated that (1) the exchange of the C-terminal 172 amino acid residues can lead to a significant decrease in both desaturase activities; (2) amino acid residues 114–174, 206–257, and 258–276 played important roles in Δ6-substrate recognition, and the last two regions were crucial for Δ8-substrate recognition; and (3) amino acid residues 114–276 of Δ6-fatty acid desaturase contained the substrate recognition site(s) responsible for discrimination between ceramide (a substrate of Δ8-sphingolipid desaturase) and acyl-PC (a substrate of Δ6-fatty acid desaturase). Substituting the amino acid residues 114-276 of RnD8A with those of RnD6C resulted in a gain of Δ6-desaturase activity in the fusion protein but a loss in Δ8-sphingolipid desaturase activity. In conclusion, several regions important for the substrate recognition of Δ8-sphingolipid desaturase and Δ6-fatty acid desaturase were identified, which provide clues in understanding the relationship between the structure and function in desaturases.

The amino acid/auxin permease (AAAP) protein family is ubiquitously present in almost all eukaryotic species and functions in various aspects of growth and development. To investigate the evolution of AAAP proteins, here 83 AAAP genes in Brassica rapa were identified, and their sequence features, and evolutionary relationships were analyzed using in silico methods. According to the phylogenetic analysis, the AAAP genes of B. rapa are divided into six clades, and these clades share relatively similar sequence features, including gene structures, conserved motifs, and domain organizations. Synteny mapping strongly suggested that segmental duplications could be responsible for the expansion of this family. Adaptive evolution analysis demonstrated that most of AAAP proteins were subject to purifying selection. However, the site Tyr257 on eight AAAP proteins from clade 2b underwent significant positive selection. Functional divergent analysis showed that type I functional divergence coefficients (θ I ) were significantly greater than zero in six pair-wise comparisons. However, functional divergence sites (Q k > 0.95) found only in the AAAP I/II and AAAP I/III comparisons were localized mainly to the trans-membrane (TM) regions, suggesting highly divergent TM structures between these groups might be associated with group-specific functions. Our results could provide a valuable clue for further investigations of the evolutionary history and biological functions of the AAAP genes in B. rapa.

Remorins play vital roles in signal transduction, energy transformation, ion flow and transport in plants. Upregulation of remorins correlates with dehiscence and cell maturation; however, no studies have been performed to elucidate the function of remorins in tree species. In this study, a Populus deltoides (Marsh.) plasma membrane-binding protein remorin gene (PdREM) was cloned and characterized by investigating its expression pattern and creating transgenic hybrid poplar (P. davidiana Dode × P. bolleana Lauche) lines expressing sense or antisense PdREM. PdREM was specifically expressed in leaf buds, and immature and mature phloem in P. deltoides. Downregulation of PdREM increased plant height, stem diameter, number of leaves, size of the xylem and phloem zones and induced expression of cell wall biosynthesis- and microfibril angle (MFA)-related genes. Overexpression of PdREM retarded vegetative growth. PdREM may negatively regulate vascular growth by inhibiting secondary cell wall expansion in poplar. In addition, antisense PdREM transgenic poplar had a lower MFA, suggesting that PdREM might contribute to sheet strength and wood properties in poplar. This study sheds light on the molecular mechanism of PdREM in P. deltoides growth and development, and lays the foundation for future functional genomics research into wood formation and the genetic engineering of forest trees with improved wood quality traits.

We elucidated the extracellular ATP (eATP) signalling cascade active in programmed cell death (PCD) using cell cultures of Populus euphratica. Millimolar amounts of eATP induced a dose- and time-dependent reduction in viability, and the agonist-treated cells displayed hallmark features of PCD. eATP caused an elevation of cytosolic Ca 2+ levels, resulting in Ca 2+ uptake by the mitochondria and subsequent H2O2 accumulation. P. euphratica exhib- ited an increased mitochondrial transmembrane potential, and cytochrome c was released without opening of the per- meability transition pore over the period of ATP stimula- tion. Moreover, the eATP-induced increase of intracellular ATP, essential for the activation of caspase-like proteases and subsequent PCD, was found to be related to increased mitochondrial transmembrane potential. NO is implicated as a downstream component of the cytosolic Ca 2+ concen- tration but plays a negligible role in eATP-stimulated cell death. We speculate that ATP binds purinoceptors in the plasma membrane, leading to the induction of downstream intermediate signals, as the proposed sequence of events in PCD signalling was terminated by the animal P2 receptor antagonist suramin.

Using confocal microscopy, X-ray microanalysis and the scanning ion-selective electrode technique, we investigated the signalling of H(2)O(2), cytosolic Ca(2+) ([Ca(2+)](cyt)) and the PM H(+)-coupled transport system in K(+)/Na(+) homeostasis control in NaCl-stressed calluses of Populus euphratica. An obvious Na(+)/H(+) antiport was seen in salinized cells; however, NaCl stress caused a net K(+) efflux, because of the salt-induced membrane depolarization. H(2)O(2) levels, regulated upwards by salinity, contributed to ionic homeostasis, because H(2)O(2) restrictions by DPI or DMTU caused enhanced K(+) efflux and decreased Na(+)/H(+) antiport activity. NaCl induced a net Ca(2+) influx and a subsequent rise of [Ca(2+)](cyt), which is involved in H(2)O(2)-mediated K(+)/Na(+) homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na(+)/H(+) antiport system, the NaCl-induced elevation of H(2)O(2) and [Ca(2+)](cyt) was correspondingly restricted, leading to a greater K(+) efflux and a more pronounced reduction in Na(+)/H(+) antiport activity. Results suggest that the PM H(+)-coupled transport system mediates H(+) translocation and triggers the stress signalling of H(2)O(2) and Ca(2+), which results in a K(+)/Na(+) homeostasis via mediations of K(+) channels and the Na(+)/H(+) antiport system in the PM of NaCl-stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.

Using the non-invasively ion-selective microelectrode technique, flux profiles of K(+), Na(+) and H(+) in mature roots and apical regions, and the effects of Ca(2+) on ion fluxes were investigated in salt-tolerant poplar species, Populus euphratica Oliver and salt-sensitive Populus simonii x (P. pyramidalis + Salix matsudana) (Populus popularis 35-44, P. popularis). Compared to P. popularis, P. euphratica roots exhibited a greater capacity to retain K(+) after exposure to a salt shock (SS, 100 mM NaCl) and a long-term (LT) salinity (50 mM NaCl, 3 weeks). Salt shock-induced K(+) efflux in the two species was markedly restricted by K(+) channel blocker, tetraethylammonium chloride, but enhanced by sodium orthovanadate, the inhibitor of plasma membrane (PM) H(+)-ATPase, suggesting that the K(+) efflux is mediated by depolarization-activated (DA) channels, e.g., KORCs (outward rectifying K(+) channels) and NSCCs (non-selective cation channels). Populus euphratica roots were more effective to exclude Na(+) than P. popularis in an LT experiment, resulting from the Na(+)/H(+) antiport across the PM. Moreover, pharmacological evidence implies that the greater ability to control K(+)/Na(+) homeostasis in salinized P. euphratica roots is associated with the higher H(+)-pumping activity, which provides an electrochemical H(+) gradient for Na(+)/H(+) exchange and simultaneously decreases the NaCl-induced depolarization of PM, thus reducing Na(+) influx via NSCCs and K(+) efflux through DA-KORCs and DA-NSCCs. Ca(2+) application markedly limited salt-induced K(+) efflux but enhanced the apparent Na(+) efflux, thus enabling the two species, especially the salt-sensitive poplar, to retain K(+)/Na(+) homeostasis in roots exposed to prolonged NaCl treatment.

The ability to rapidly switch the intracellular energy storage form from starch to lipids is an advantageous trait for microalgae feedstock. To probe this mechanism, we sequenced the 56.8-Mbp genome of Chlorella pyrenoidosa FACHB-9, an industrial production strain for protein, starch, and lipids. The genome exhibits positive selection and gene family expansion in lipid and carbohydrate metabolism and genes related to cell cycle and stress response. Moreover, 10 lipid metabolism genes might be originated from bacteria via horizontal gene transfer. Transcriptomic dynamics tracked via messenger RNA sequencing over six time points during metabolic switch from starch-rich heterotrophy to lipid-rich photoautotrophy revealed that under heterotrophy, genes most strongly expressed were from the tricarboxylic acid cycle, respiratory chain, oxidative phosphorylation, gluconeogenesis, glyoxylate cycle, and amino acid metabolisms, whereas those most down-regulated were from fatty acid and oxidative pentose phosphate metabolism. The shift from heterotrophy into photoautotrophy highlights up-regulation of genes from carbon fixation, photosynthesis, fatty acid biosynthesis, the oxidative pentose phosphate pathway, and starch catabolism, which resulted in a marked redirection of metabolism, where the primary carbon source of glycine is no longer supplied to cell building blocks by the tricarboxylic acid cycle and gluconeogenesis, whereas carbon skeletons from photosynthesis and starch degradation may be directly channeled into fatty acid and protein biosynthesis. By establishing the first genetic transformation in industrial oleaginous C. pyrenoidosa, we further showed that overexpression of an NAD(H) kinase from Arabidopsis (Arabidopsis thaliana) increased cellular lipid content by 110.4%, yet without reducing growth rate. These findings provide a foundation for exploiting the metabolic switch in microalgae for improved photosynthetic production of food and fuels.

To improve the transformation efficiency of wheat (Triticum aestivum L.) mediated by Agrobacterium tumefaciens, we explored the possibility of employing the basal portion of wheat seedling (shoot apical meristem) as the explants. Three genotypes of wheat were transformed by A. tumefaciens carrying beta-1, 3-glucanase gene. After vernalization, the seeds to be transformed were germinated. When these seedlings grew up to 2 approximately 5 cm, their coleoptile and half of the cotyledon were cut out, and the basal portions were infected by A. tumefaciens. A total 27 T(0) transgenic plants were obtained, and the average transformation efficiency was as high as 9.82%. Evident segregation occurred in some of the T(1) plants, as was indicated by PCR and Southern blotting analysis. Investigation of the T(2) plants revealed that some transformed plants had higher resistance to powdery mildew than the controls. Northern blotting revealed that beta-1, 3-glucanase gene was normally expressed in the T(2) plants, which showed an increased resistance to powdery mildew. The results above indicate that the exogenous gene has been successfully integrated into the genome of wheat, transmitted and expressed in the transgenic progeny. From all the results above, it can be concluded that Agrobacterium inoculum to the basal portion of wheat seedling is a highly efficient and dependable transformation method. It can be developed into a practicable method for transfer of target gene into wheat.

Chromosome 1B was microdissected and collected from chromosome spreads of wheat (Triticum aestivum L. ‘Jing 411’), using a glass needle. DNA of the isolated chromosome was amplified in vitro by Sau3A linker adaptor-mediated polymerase chain reaction (LA-PCR). The second-round PCR products were verified by Southern hybridization using DIG-labeled genomic DNA of wheat. The results initially showed the DNA was from wheat genome. A pair of SSR primers specific to chromosome 1B was used to verify the origin of the PCR products from the isolated chromosome. The results confirmed that the PCR products originated from chromosome 1B. The second round of PCR products from chromosome 1B were cloned into plasmid pUCm-T vectors to create a chromosome-specific library, which included approximately 248 000 recombinant clones. Characterization of 100 randomly selected clones of the library showed that the insert size ranged between 0.5 and 2.0 kb, with an average of 1 kb. Randomly selected 288 clones were characterized with dot blot hybridization, of which 57.2% were medium/high copy clones and 42.8% low/single copy clones. The application of this technique to establish high-density molecular maps for chromosome 1B is discussed. Key words: Wheat, chromosome microdissection, chromosome-specific library

Summary Low-temperature soluble carbohydrate accumulations are commonly associated with anthocyanin coloration, attenuated growth, and cold adaptation of cool-season grasses. A total of 647 AFLP markers were tested for associations with anthocyanin coloration, tiller formation, leaf formation, cumulative leaf length, percent soluble carbohydrate, and dry matter regrowth among replicated clones of an admixed Leymus wildrye breeding population evaluated in low-temperature growth chambers. The admixed breeding population was derived from a heterogeneous population of L. cinereus × L. triticoides F1 hybrids, with two additional generations of open pollination. Two AFLP linkage maps, constructed from two full-sib mapping populations derived from the same F1 hybrid population, were integrated to produce a framework consensus map used to examine the distribution of marker-trait associations in the admixed F1OP2 population. Thirty-seven linkage blocks, spanning 258 cM (13.6%) of the 1895 cM consensus map, contained 119 (50%) of the 237 markers showing at least one possible trait association (P < 0.05). Moreover, 28 (68%) of the 41 most significant marker-trait associations (P < 0.005) were located in 15 QTL linkage blocks spanning 112.9 cM (6%) of the linkage map. The coincidence of these 28 significant marker-trait associations, and many less significant associations, in 15 relatively small linkage blocks (0.6 cM to 21.3 cM) provides evidence of admixture linkage disequilibrium QTLs (ALD QTLs) in this heterogeneous breeding population. At least four of the remaining 13 putative marker-trait associations (P < 0.005) were located in genetic map regions lacking other informative markers. The complexity of marker-trait associations results from heterogeneity within and substantial divergence among the parental accessions. Abbreviations: ALD ‐ admixture linkage disequilibrium; CHO ‐ carbohydrate

Three Triticum aestivum L. × Thinopyrum junceum (L.) A. Love partial amphidiploids (2n=8x=56; 21 \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage[OT2,OT1]{fontenc} \newcommand\cyr{ \renewcommand\rmdefault{wncyr} \renewcommand\sfdefault{wncyss} \renewcommand\encodingdefault{OT2} \normalfont \selectfont} \DeclareTextFontCommand{\textcyr}{\cyr} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \landscape $\arcsec$\end{document} ABD + 7 \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage[OT2,OT1]{fontenc} \newcommand\cyr{ \renewcommand\rmdefault{wncyr} \renewcommand\sfdefault{wncyss} \r...

Hycrest is an outcrossing tetraploid cultivar of crested wheatgrass developed from a hybrid between an induced tetraploid form of Fairway (Agropyron cristatum) and natural tetraploid Standard (A. desertorum). The CD-II cultivar was selected from cv. Hycrest on the basis of vigorous vegetative growth and green leaf coloration during early spring. This study examines the selection response of molecular markers associated with anthocyanin coloration (AC), growth habit (GH) and other traits in Hycrest, CD-II, and two second-generation polycross (PX2) populations derived from three purple-leaf selections and three green-leaf selections of Hycrest. AC was positively correlated with prostrate GH and inversely correlated with plant height and leaf width in the experimental PX2 populations. Of the 578 AFLP markers surveyed, 13 showed pleiotropic effects on GH and AC in the PX2 populations. In all cases, marker alleles associated with prostrate GH also enhanced AC. Four of these 13 markers also showed large selection responses in CD-II (i.e. 80th percentile or higher) relative to Hycrest, always favouring upright GH and low AC alleles.However, the upright GH in CD-II was not explainable solely by molecular marker alleles near the vernalization gene. Key words: Anthocyanin, crested wheatgrass, molecular marker, selection, vernalization

A single chromosome was microdissected from the metaphase chromosomes of soybean by using a glass needle. After two rounds of Sau3A linker-adaptor-mediated PCR, smear DNA fragments ranged from 0.3–2.5 kb were obtained. Southern hybridization showed that the PCR products from the dissected chromosome were homologous with the soybean genomic DNA. In situ hybridization of the chromosome PCR products to the mitotic metaphase spreads showed that the PCR products were hybridized to the soybean, but the chromosomal signals were not restricted to the microdissected chromosome. The second-round PCR products from the microdissected chromosome were cloned into plasmid vector to generate a chromosome-specific microclone library, which included approximately 200,000 recombinant clones. 178 randomly selected clones were estimated to range in size from 0.3∼1.8 kb, with an average size of 830 bp, of which approximately 44% were high/medium copy clones, while 56% were low/unique copy clones.

Populus euphratica is a salt-tolerant tree species that develops leaf succulence after a prolonged period of salinity stress. In the present study, a putative xyloglucan endotransglucosylase/hydrolase gene (PeXTH) from P. euphratica was isolated and transferred to tobacco plants. PeXTH localized exclusively to the endoplasmic reticulum and cell wall. Plants overexpressing PeXTH were more salt tolerant than wild-type tobacco with respect to root and leaf growth, and survival. The increased capacity for salt tolerance was due mainly to the anatomical and physiological alterations caused by PeXTH overexpression. Compared with the wild type, PeXTH-transgenic plants contained 36% higher water content per unit area and 39% higher ratio of fresh weight to dry weight, a hallmark of leaf succulence. However, the increased water storage in the leaves in PeXTH-transgenic plants was not accompanied by greater leaf thickness but was due to highly packed palisade parenchyma cells and fewer intercellular air spaces between mesophyll cells. In addition to the salt dilution effect in response to NaCl, these anatomical changes increased leaf water-retaining capacity, which lowered the increase of salt concentration in the succulent tissues and mesophyll cells. Moreover, the increased number of mesophyll cells reduced the intercellular air space, which improved carbon economy and resulted in a 47-78% greater net photosynthesis under control and salt treatments (100-150 mM NaCl). Taken together, the results indicate that PeXTH overexpression enhanced salt tolerance by the development of succulent leaves in tobacco plants without swelling. peerReviewed

Commercial and non-commercial plants face a variety of environmental stressors that often cannot be controlled. In this study, transgenic hybrid poplar (Populus × euramericana 'Guariento') harboring five effector genes (vgb, SacB, JERF36, BtCry3A and OC-I) were subjected to drought, salinity, waterlogging and insect stressors in greenhouse or laboratory conditions. Field trials were also conducted to investigate long-term effects of transgenic trees on insects and salt tolerance in the transformants. In greenhouse studies, two transgenic lines D5-20 and D5-21 showed improved growth, as evidenced by greater height and basal diameter increments and total biomass relative to the control plants after drought or salt stress treatments. The improved tolerance to drought and salt was primarily attributed to greater instantaneous water use efficiency (WUEi) in the transgenic trees. The chlorophyll concentrations tended to be higher in the transgenic lines under drought or saline conditions. Transformed trees in drought conditions accumulated more fructan and proline and had increased Fv/Fm ratios (maximum quantum yield of photosystem II) under waterlogging stress. Insect-feeding assays in the laboratory revealed a higher total mortality rate and lower exuviation index of leaf beetle [Plagiodera versicolora (Laicharting)] larvae fed with D5-21 leaves, suggesting enhanced insect resistance in the transgenic poplar. In field trials, the dominance of targeted insects on 2-year-old D5-21 transgenic trees was substantially lower than that of the controls, indicating enhanced resistance to Coleoptera. The average height and DBH (diameter at breast height) of 2.5-year-old transgenic trees growing in naturally saline soil were 3.80% and 4.12% greater than those of the control trees, but these increases were not significant. These results suggested that multiple stress-resistance properties in important crop tree species could be simultaneously improved, although additional research is needed to fully understand the relationships between the altered phenotypes and the function of each transgene in multigene transformants.

Gamma-linolenic acid (gamma-linolenic acid, GLA; C18:3 Delta(6, 9, 12)) belongs to the omega-6 family and exists primarily in several plant oils, such as evening primrose oil, blackcurrant oil, and borage oil. Delta(6)-desaturase is a key enzyme involved in the synthesis of GLA. There have been no previous reports on the genes encoding Delta(6)-desaturase in blackcurrant (Ribes nigrum L.). In this research, five nearly identical copies of Delta(6)-desaturase gene-like sequences, named RnD8A, RnD8B, RnD6C, RnD6D, and RnD6E, were isolated from blackcurrant. Heterologous expression in Saccharomyces cerevisiae and/or Arabidopsis thaliana confirmed that RnD6C/D/E were Delta(6)-desaturases that could use both alpha-linolenic acids (ALA; C18:3 Delta(9,12,15)) and linoleic acid (LA; C18:2 Delta(9,12)) precursors in vivo, whereas RnD8A/B were Delta(8)-sphingolipid desaturases. Expression of GFP tagged with RnD6C/D/E showed that blackcurrant Delta(6)-desaturases were located in the mitochondrion (MIT) in yeast and the endoplasmic reticulum (ER) in tobacco. GC-MS results showed that blackcurrant accumulated GLA and octadecatetraenoic acids (OTA; C18:4 Delta(6,9,12,15)) mainly in seeds and a little in other organs and tissues. RT-PCR results showed that RnD6C and RnD6E were expressed in all the tissues at a low level, whereas RnD6D was expressed at a high level only in seeds, leading to the accumulation of GLA and OTA in seeds. This research provides new insights to our understanding of GLA synthesis and accumulation in plants and the evolutionary relationship of this class of desaturases, and new clues as to the amino acid determinants which define precise enzyme activity.

Using the Affymetrix poplar genome array, we explored the leaf transcriptome of salt-tolerant Populus euphratica Oliv. and salt-sensitive P. popularis 35-44 (P. popularis) under control and saline conditions. Our objective was to clarify the genomic differences in regulating K(+)/Na(+) and reactive oxygen species (ROS) homeostasis between the two species. Compared to P. popularis, salt-tolerant P. euphratica responses to salinity involved induction of a relatively larger number of probesets after short-term (ST) exposure to 150 mM NaCl (24 h) and relatively fewer probesets after a long-term (LT) exposure to salinity (200 mM NaCl, 28 days). Compared to P. popularis, leaves of the control P. euphratica plants exhibited a higher transcript abundance of genes related to Na(+)/H(+) antiport (Na(+)/H(+) antiporters, H(+) pumps) and K(+) uptake and transport. Notably, the expression of these genes did not decrease (with a few exceptions) during salt treatment. Regarding ROS homeostasis, P. euphratica exhibited rapid up-regulation of a variety of antioxidant enzymes after exposure to ST salinity, indicating a rapid adaptive response to salt stress. However, the effect of NaCl on transcription in P. popularis leaves was more pronounced after exposure to prolonged salinity. LT-stressed P. popularis up-regulated some genes mediating K(+)/Na(+) homeostasis but decreased transcription of main scavengers of superoxide radicals and H(2)O(2) except for some isoforms of a few scavengers. Mineral and ROS analyses show that NaCl induced a marked increase of leaf Na(+) and H(2)O(2) in LT-stressed plants of the two species and the effects were even more pronounced in the salt-sensitive poplar. We place the transcription results in the context of our physiological measurements to infer some implications of NaCl-induced alterations in gene expression related to K(+)/Na(+) and ROS homeostasis.

The ability of a plant to maintain an ionic homeostasis is crucial in plant salt tolerance. Direct evidence based on data from the non-invasive measurement of ion fluxes would not only offer new insight about the function of the transporter but also provide a whole plant approach for dissecting salt adaptation mechanisms. Here, we review some reports using the ion-selective microelectrodes to characterize the net ion fluxes of tissues or cells.

Background To obtain important expressed sequence tags (ESTs) located on specific chromosomes is currently difficult. Construction of single-chromosome EST library could be an efficient strategy to isolate important ESTs located on specific chromosomes. In this research we developed a method to rapidly isolate ESTs from chromosome 1R of rye by combining the techniques of chromosome microdissection with hybrid specific amplification (HSA). Results Chromosome 1R was isolated by a glass needle and digested with proteinase K (PK). The DNA of chromosome 1R was amplified by two rounds of PCR using a degenerated oligonucleotide 6-MW sequence with a Sau3AI digestion site as the primer. The PCR product was digested with Sau3AI and linked with adaptor HSA1, then hybridized with the Sau3AI digested cDNA with adaptor HSA2 of rye leaves with and without salicylic acid (SA) treatment, respectively. The hybridized DNA fragments were recovered by the HSA method and cloned into pMD18-T vector. The cloned inserts were released by PCR using the partial sequences in HSA1 and HSA2 as the primers and then sequenced. Of the 94 ESTs obtained and analyzed, 6 were known sequences located on rye chromosome 1R or on homologous group 1 chromosomes of wheat; all of them were highly homologous with ESTs of wheat, barley and/or other plants in Gramineae, some of which were induced by abiotic or biotic stresses. Isolated in this research were 22 ESTs with unknown functions, probably representing some new genes on rye chromosome 1R. Conclusion We developed a new method to rapidly clone chromosome-specific ESTs from chromosome 1R of rye. The information reported here should be useful for cloning and investigating the new genes found on chromosome 1R.

Background Oil in the form of triacylglycerols (TAGs) is quantitatively the most important storage form of energy for eukaryotic cells. Diacylglycerol acyltransferase (DGAT) is considered the rate-limiting enzyme for TAG accumulation. Chlorella, a unicellular eukaryotic green alga, has attracted much attention as a potential feedstock for renewable energy production. However, the function of DGAT1 in Chlorella has not been reported. Results A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Chlorella ellipsoidea. The 2,142 bp open reading frame of this cDNA, designated CeDGAT1, encodes a protein of 713 amino acids showing no more than 40% identity with DGAT1s of higher plants. Transcript analysis showed that the expression level of CeDGAT1 markedly increased under nitrogen starvation, which led to significant triacylglycerol (TAG) accumulation. CeDGAT1 activity was confirmed in the yeast quadruple mutant strain H1246 by restoring its ability to produce TAG. Upon expression of CeDGAT1, the total fatty acid content in wild-type yeast (INVSc1) increased by 142%, significantly higher than that transformed with DGAT1s from higher plants, including even the oil crop soybean. The over-expression of CeDGAT1 under the NOS promoter in wild-type Arabidopsis thaliana and Brassica napus var. Westar significantly increased the oil content by 8–37% and 12–18% and the average 1,000-seed weight by 9–15% and 6–29%, respectively, but did not alter the fatty acid composition of the seed oil. The net increase in the 1,000-seed total lipid content was up to 25–50% in both transgenic Arabidopsis and B. napus. Conclusions We identified a gene encoding DGAT1 in C. ellipsoidea and confirmed that it plays an important role in TAG accumulation. This is the first functional analysis of DGAT1 in Chlorella. This information is important for understanding lipid synthesis and accumulation in Chlorella and for genetic engineering to enhance oil production in microalgae and oil plants. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-0995-5) contains supplementary material, which is available to authorized users.