MicroRNAs (miRNAs) are small, non-coding RNAs that play essential roles in plant growth, development, and stress response. Populus euphratica is a typical abiotic stress-resistant woody species. This study presents an efficient method for genome-wide discovery of new drought stress responsive miRNAs in P. euphratica. High-throughput sequencing of P. euphratica leaves found 197 conserved miRNAs between P. euphratica and Populus trichocarpa. Meanwhile, 58 new miRNAs belonging to 38 families were identified, an increase in the number of P. euphratica miRNAs. Twenty-six new and 21 conserved miRNA targets were verified by degradome sequencing, and target annotation showed that these targets were involved in multiple biological processes, including transcriptional regulation and response to stimulus. Furthermore, comparison of high-throughput sequencing with miRNA microarray profiling data indicated that 104 miRNA sequences were up-regulated, whereas 27 were down-regulated under drought stress. This preliminary characterization provides a framework for future analysis of miRNA genes and their roles in key poplar traits such as stress resistance, and could be useful for plant breeding and environmental protection

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263 SummaryPollen-and seed-mediated transgene flow is a concern in plant biotechnology. We report here a highly efficient 'genetically modified (GM)-gene-deletor' system to remove all functional transgenes from pollen, seed or both. With the three pollen-and / or seed-specific gene promoters tested, the phage CRE/ loxP or yeast FLP/ FRT system alone was inefficient in excising transgenes from tobacco pollen and/or seed, with no transgenic event having 100% efficiency. When loxP-FRT fusion sequences were used as recognition sites, simultaneous expression of both FLP and CRE reduced the average excision efficiency, but the expression of FLP or CRE alone increased the average excision efficiency, with many transgenic events being 100% efficient based on more than 25 000 T 1 progeny examined per event. The 'GM-gene-deletor' reported here may be used to produce 'non-transgenic' pollen and/or seed from transgenic plants and to provide a bioconfinement tool for transgenic crops and perennials, with special applicability towards vegetatively propagated plants and trees.

Here we describe a simple and rapid method for assembly and PCR-based accurate synthesis (PAS) of long DNA sequences. The PAS protocol involves the following five steps: (i) design of the DNA sequence to be synthesized and of 60-bp overlapping oligonucleotides to cover the entire DNA sequence; (ii) purification of the oligonucleotides by PAGE; (iii) first PCR, to synthesize DNA fragments of 400-500 bp in length using 10 inner (template) and two outer (primer) oligonucleotides; (iv) second PCR, to assemble the products of the first PCR into the full-length DNA sequence; and (v) cloning and verification of the synthetic DNA by sequencing and, if needed, error correction using an overlap-extension PCR technique. This method, which takes approximately 1 wk, is suitable for synthesizing diverse types of long DNA molecule. We have successfully synthesized DNA fragments from 0.5 to 12.0 kb, with high G+C content, repetitive sequences or complex secondary structures. The PAS protocol therefore provides a simple, rapid, reliable and relatively inexpensive method for synthesizing long, accurate DNA sequences.

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The CYP74B2 gene in Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col) contains a 10-nucleotide deletion in its first exon that causes it to code for a truncated protein not containing the P450 signature typical of other CYP74B subfamily members. Compared to CYP74B2 transcripts in the Landsberg erecta (Ler) ecotype that code for full-length hydroperoxide lyase (HPL) protein, CYP74B2 transcripts in the Col ecotype accumulate at substantially reduced levels. Consistent with the nonfunctional HPL open reading frame in the Col ecotype, in vitro HPL activity analyses using either linoleic acid hydroperoxide or linolenic acid hydroperoxide as substrates show undetectable HPL activity in the Col ecotype and C6 volatile analyses using leaf homogenates show substantially reduced amounts of hexanal and no detectable trans-2-hexenal generated in the Col ecotype. P450-specific microarrays and full-genome oligoarrays have been used to identify the range of other transcripts expressed at different levels in these two ecotypes potentially as a result of these variations in HPL activity. Among the transcripts expressed at significantly lower levels in Col leaves are those coding for enzymes involved in the synthesis of C6 volatiles (LOX2, LOX3), jasmonates (OPR3, AOC), and aliphatic glucosinolates (CYP83A1, CYP79F1, AOP3). Two of the three transcripts coding for aliphatic glucosinolates (CYP83A1, AOP3) are also expressed at significantly lower levels in Col flowers.

Populus euphratica, a typical hydro-halophyte, is ideal for studying salt stress responses in woody plants. MicroRNAs (miRNAs) are endogenous non-coding small RNAs that fulfilled an important post-transcriptional regulatory function. MiRNA may regulate tolerance to salt stress but this has not been widely studied in P. euphratica. In this investigation, the small RNAome, degradome and transcriptome were studied in salt stress treated P. euphratica by deep sequencing. Two hundred and eleven conserved miRNAs between Populus trichocarpa and P. euphratica have been found. In addition, 162 new miRNAs, belonging to 93 families, were identified in P. euphratica. Degradome sequencing experimentally verified 112 targets that belonged to 51 identified miRNAs, few of which were known previously in P. euphratica. Transcriptome profiling showed that expression of 15 miRNA-target pairs displayed reverse changing pattern under salt stress. Together, these results indicate that, in P. euphratica under salt stress, a large number of new miRNAs could be discovered, and both known and new miRNA were functionally cleaving to their target mRNA. Expression of miRNA and target were correspondingly induced by salt stress but that it was a complex process in P. euphratica.

Here we report the effect of the 35S promoter sequence on activities of the tissue- and organ-specific gene promoters in tobacco plants. In the absence of the 35S promoter sequence the AAP2 promoter is active only in vascular tissues as indicated by expression of the AAP2:GUS gene. With the 35S promoter sequence in the same T-plasmid, transgenic plants exhibit twofold to fivefold increase in AAP2 promoter activity and the promoter becomes active in all tissue types. Transgenic plants hosting the ovary-specific AGL5:iaaM gene (iaaM coding an auxin biosynthetic gene) showed a wild-type phenotype except production of seedless fruits, whereas plants hosting the AGL5:iaaM gene along with the 35S promoter sequence showed drastic morphological alterations. RT-PCR analysis confirms that the phenotype was caused by activation of the AGL5:iaaM gene in non-ovary organs including roots, stems and flowers. When the pollen-, ovule- and early embryo-specific PAB5:barnase gene (barnase coding a RNase gene) was transformed, the presence of 35S promoter sequence drastically reduced transformation efficiencies. However, the transformation efficiencies were restored in the absence of 35S promoter, indicating that the 35S promoter might activate the expression of PAB5:barnase in non-reproductive organs such as calli and shoot primordia. Furthermore, if the 35S promoter sequence was replaced with the NOS promoter sequence, no alteration in AAP2, AGL5 or PAB5 promoter activities was observed. Our results demonstrate that the 35S promoter sequence can convert an adjacent tissue- and organ-specific gene promoter into a globally active promoter.