MIR166a Affects the Germination of Somatic Embryos in <Emphasis Type="Italic">Larixleptolepis</Emphasis> by Modulating IAA Biosynthesis and Signaling Genes

The somatic embryogenic regeneration system is an ideal model system to study the regulation of early developmental processes and morphogenesis in gymnosperms. We have previously generated five larch (Larix leptolepis) LaMIR166a overexpression cell lines. The germination rates of mature somatic embryos in transgenic and wild-type (WT) lines were calculated and the results showed that overexpression of the miR166a precursor (LaMIR166a) markedly enhanced germination, especially in the a-3, a-4, and a-5 lines. The relative expression of LaMIR166a and miR166a in the LaMIR166a overexpression lines was higher than in the WT control line during the germination process, whereas the expression levels of LaHDZ31–34 increased markedly throughout germination, potentially as a result of feedback regulation of miR166. The effect of miR166a on auxin biosynthesis and signaling genes was also studied. During germination, mRNA levels of Nitrilase (LaNIT), Auxin response factor1 (LaARF1), and LaARF2 were markedly higher in LaMIR166a overexpressing lines. These results indicated that indole-3-acetic acid (IAA) synthesis is required for germination in L. leptolepis. Further exogenous application of IAA at different concentrations showed that 2 mg L^?1 IAA clearly promoted germination, resulting in a 56% germination rate for L. leptolepis somatic embryos. This shows that IAA plays a vital role in controlling the germination ability of someatic embryos in L. leptolepis. Our results suggest that miR166a and LaHDZ31–34 have important roles in auxin biosynthesis and signaling during the germination of somatic embryos in L. leptolepis.     

Overexpression of a <Emphasis Type="Italic">Populus euphratica CBF4</Emphasis> gene in poplar confers tolerance to multiple stresses

C-repeat binding factors (CBFs) play a key role in abiotic stresses. However, little is known about CBFs in Populus euphratica. Here, we isolated PeCBF4a, a member of CBF gene family from P. euphratica. Its expression was induced by dehydration, salinity and low temperature. We generated transgenic poplars (Populus tomentosa ‘YiXianCiZhu B385’) overexpressing PeCBF4a (OE-PeCBF4a) under the control of the CaMV 35S promoter or transformed with empty vector. The wild-type (WT) and empty vector lines were used as controls. Under abiotic stresses, the photosynthetic rate (Pn) of 60-day-old OE-PeCBF4a lines increased 34.7–165.7?% and the instantaneous water use efficiency (iWUE) increased 48.9–103.7?% over controls. The maximum quantum efficiency of PSII photochemistry (Fv/Fm) values in PeCBF4a-overexpressing lines did not change significantly and were 2.14–5.89?% higher. The non-photochemical quenching coefficient (NPQ) mean of OE-PeCBF4a lines decreased by 12.02–23.64?% while the photochemical quenching (qP) value was 8.75–22.31?% higher than controls. OE-PeCBF4a lines also displayed higher superoxide dismutase (SOD) activities and markedly lower malondialdehyde (MDA) levels compared to controls. Higher levels of proline and sugars accumulated in transgenic plants. Overexpression of PeCBF4a not only induced strong expression of the stress-responsive downstream target genes of PeCBF4a, PtRCI2A (rare-cold-inducible 2A) and PtDI21 (drought-induced 21), but also caused dwarfed phenotypes. Based on results from P-V curve measurements, the osmotic adjustment capability of OE-PeCBF4a plants was enhanced. These results confirmed that OE-PeCBF4a poplars exhibit greater tolerance to stress, indicating that PeCBF4a plays a positive role in stress tolerance.     

Cucumber <Emphasis Type="Italic">Phospholipase D alpha</Emphasis> gene overexpression in tobacco enhanced drought stress tolerance by regulating stomatal closure and lipid peroxidation

Background

Plant phospholipase D (PLD), which can hydrolyze membrane phospholipids to produce phosphatidic acid (PA), a secondary signaling molecule, has been proposed to function in diverse plant stress responses. Both PLD and PA play key roles in plant growth, development, and cellular processes. PLD was suggested to mediate the regulation of stomatal movements by abscisic acid (ABA) as a response to water deficit. In this research, we characterized the roles of the cucumber phospholipase D alpha gene (CsPLDα, GenBank accession number EF363796) in the growth and tolerance of transgenic tobacco (Nicotiana tabacum) to drought stress.

Results

The CsPLDα overexpression in tobacco lines correlated with the ABA synthesis and metabolism, regulated the rapid stomatal closure in drought stress, and reduced the water loss. The NtNCED1 expression levels in the transgenic lines and wild type (WT) were sharply up-regulated after 16?days of drought stress compared with those before treatment, and the expression level in the transgenic lines was significantly higher than that in the WT. The NtAOG expression level evidently improved after 8 and 16?days compared with that at 0?day of treatment and was significantly lower in the transgenic lines than in the WT. The ABA content in the transgenic lines was significantly higher than that in the WT. The CsPLDα overexpression could increase the osmolyte content and reduce the ion leakage. The proline, soluble sugar, and soluble protein contents significantly increased. By contrast, the electrolytic leakage and malondialdehyde accumulation in leaves significantly decreased. The shoot and root fresh and dry weights of the overexpression lines significantly increased. These results indicated that a significant correlation between CsPLDα overexpression and improved resistance to water deficit.

Conclusions

The plants with overexpressed CsPLDα exhibited lower water loss, higher leaf relative water content, and heavier fresh and dry matter accumulation than the WT. We proposed that CsPLDα was involved in the ABA-dependent pathway in mediating the stomatal closure and preventing the elevation of intracellular solute potential.

     

Constitutive expression of a plant ferredoxin-like protein (pflp) enhances capacity of photosynthetic carbon assimilation in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

The plant ferredoxin-like protein (PFLP) gene, cloned from sweet peppers predicted as an electron carrier in photosynthesis, shows high homology to the Fd-I sequence of Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Spinacia oleracea. Most of pflp related studies focused on anti-pathogenic effects, while less understanding for the effects in photosynthesis with physiological aspects, such as photosynthesis rate, and levels of carbohydrate metabolites. This project focuses on the effects of pflp overexpression on photosynthesis by physiological evaluations of carbon assimilation with significant higher levels of carbohydrates with higher photosynthesis efficiency. In this report, two independent transgenic lines of rice plants (designated as pflp-1 and pflp-2) were generated from non-transgenic TNG67 rice plant (WT). Both transgenic pflp rice plants exhibited enhanced photosynthesis efficiency, and gas exchange rates of photosynthesis were 1.3- and 1.2-fold higher for pflp-1 and pflp-2 than WT respectively. Significantly higher electron transport rates of pflp rice plants were observed. Moreover, photosynthetic products, such as fructose, glucose, sucrose and starch contents of pflp transgenic lines were increased accordingly. Molecular evidences of carbohydrate metabolism related genes activities (osHXK5, osHXK6, osAGPL3, osAGPS2α, osSPS, ospFBPase, oscFBPase, and osSBPase) in transgenic lines were higher than those of WT. For performance of crop production, 1000-grain weight for pflp-1 and pflp-2 rice plants were 52.9 and 41.1 g that were both significantly higher than 31.6 g for WT, and panicles weights were 1.4- and 1.2-fold higher than WT. Panicle number, tiller number per plants for pflp rice plants were all significantly higher compared with those of WT where there was no significant difference observed between two pflp rice plants. Taken altogether; this study demonstrated that constitutive pflp expression can improve rice production by enhancing the capacity of photosynthetic carbon assimilation.     

Effects of the maize C<Subscript>4</Subscript> phosphoenolpyruvate carboxylase (<Emphasis Type="Italic">ZmPEPC</Emphasis>) gene on nitrogen assimilation in transgenic wheat

Nitrogen (N) is the primary limiting factor for crop growth, development, and productivity. Transgenic technology is a straightforward strategy for improving N assimilation in crops. The present study assessed the effects of maize C₄ phosphoenolpyruvate carboxylase (ZmPEPC) gene overexpression on N assimilation in three independent transgenic lines and wild-type (WT) wheat (Triticum aestivum L.). The transgenic wheat lines depicted ZmPEPC overexpression and higher PEPC enzyme activity relative to that in the WT. The leaves of the transgenic wheat lines subjected to low N treatment showed an increase in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) expression, content, and carboxylase activity. The transgenic wheat lines also depicted an upregulation of genes associated with the anaplerotic pathway for the TCA cycle, suggesting that more carbon (C) skeleton material is being allocated for N assimilation under low N conditions. Furthermore, ZmPEPC expression in transgenic wheat lines induced the upregulated of genes associated primary N metabolism, including TaNR, TaGS2, TaGOGAT, TaAspAT, and TaASN1. The average total free amino acid content in the transgenic wheat lines was 48.18% higher than that in the WT, and asparagine (Asn), glutamine (Gln), aspartic acid (Asp), and serine (Ser) were also markedly enhanced. In addition, elementary analysis showed that N and C content, and the biomass of the transgenic wheat lines increased with low N treatment. Yield trait analysis indicated that ZmPEPC overexpression improved grain yield by increasing 1000-grain weight. In conclusion, ZmPEPC overexpression in wheat could modulate C metabolism, significantly improve N assimilation, enhances growth, and improves yield under low N conditions.     

Overexpression of Loquat Dehydrin Gene <Emphasis Type="Italic">EjDHN1</Emphasis> Promotes Cold Tolerance in Transgenic Tobacco

Dehydrins (DHNs) play vital roles in response to dehydration stress in plants. To examine the contribution of EjDHN to low-temperature stress in loquat (Eriobotrya japonica Lindl.), EjDHN1 was overexpressed in tobacco (Nicotiana tabacum L.). The plant growth of transgenic lines was significantly better than wild type (WT) after 4 d of recovery from cold stress. Cold stress led to membrane lipid peroxidation and reduced photosystem II (PSII) activity in leaves, and these were less severe in transgenic lines. To examine oxidative stress tolerance, the plants were treated with different concentrations of methyl viologen (MV), which inhibited plant growth both in WT and transgenic lines. After exposure to 2.0 μM MV for 10 d, the WT plants had a dramatically lower survival rate. MV treatment in leaf disks confirmed that transgenic lines accumulated less reactive oxygen species (ROS) and suffered less lipid peroxidation. The results suggested that the tolerance of the transgenic plants to cold was increased, and EjDHN1 could protect cells against oxidative damage caused by ROS production under cold stress. It also provided evidences that the enhanced cold tolerance resulted from EjDHN1 overexpression could be partly due to their protective effect on membranes by alleviating oxidative stresses.     

Novel constructs for efficient cloning of sRNA-encoding DNA and uniform silencing of plant genes employing artificial <Emphasis Type="Italic">trans</Emphasis>-acting small interfering RNA

Key message

TAS atasiRNA-producing region swapping used one-step, high efficiency, and high fidelity directional TC-cloning. Uniform silencing was achieved without lethality using miRNA trigger- TAS overexpression fusion cassettes to generate 21-nt atasiRNA.

Abstract

Plant transgenic technologies are very important for basic plant research and biotechnology. Artificial trans-acting small interfering RNA (atasiRNA) represents an attractive platform with certain advantages over other silencing approaches, such as hairpin RNA, artificial microRNA (amiRNA), and virus-induced gene silencing (VIGS). In this study, we developed two types of constructs for atasiRNA-mediated gene silencing in plants. To functionally validate our constructs, we chose TAS1a as a test model. Type 1 constructs had miR173-precursor sequence fused with TAS1a locus driven by single promoter–terminator cassette, which simplified the expression cassette and resulted in uniform gene silencing. Type 2 constructs contained two separate cassettes for miR173 and TAS1a co-expression. The constructs in each type were further improved by deploying the XcmI-based TC-cloning system for highly efficient directional cloning of short DNA fragments encoding atasiRNAs into TAS1a locus. The effectiveness of the constructs was demonstrated by cloning an atasiRNA DNA into the TC site of engineered TAS1a and silencing of CHLORINA 42 (CH42) gene in Arabidopsis. Our results show that the directional TC-cloning of the atasiRNA DNA into the engineered TAS1a is highly efficient and the miR173–TAS1a fusion system provides an attractive alternative to achieve moderate but more uniform gene silencing without lethality, as compared to conventional two separate cassettes for miR173 and TAS locus co-expression system. The design principles described here should be applicable to other TAS loci such as TAS1b, TAS1c, TAS2, or TAS3, and cloning of amiRNA into amiRNA stem-loop.

     

Functional Conservation of an <Emphasis Type="Italic">AGAMOUS</Emphasis> Orthologous Gene Controlling Reproductive Organ Development in the Gymnosperm Species <Emphasis Type="Italic">Taxus chinensis</Emphasis> var. <Emphasis Type="Italic">mairei</Emphasis>

Arabidopsis AGAMOUS (AG) has roles in specifying reproductive organ (stamens and carpels) identity, floral meristem determinacy, and repression of A-function. To investigate possible roles of AG orthologous genes in gymnosperm species and evolution of C function, we isolated and identified AG orthologous gene TcAG from Taxus chinensis var. mairei (family Taxaceae, order Coniferales), a member of the last divergant lineage from higher Conifer that sisters to Gnetales. Sequence alignment and phylogenetic analysis grouped TcAG into the gymnosperm AG lineage. TcAG was expressed in both developing male and female cones, but there was no expression in juvenile leaves. Ectopic expression of TcAG in an Arabidopsis ag mutant produced flowers with the third whorl petaloid stamen and fourth whorl normal carpel, but failed to convert first whorl sepals into carpeloid organs and second whorl petals into stamenoid organs. A 35S::TcAG transgenic Arabidopsis ag mutant had very early flowering, and produced a misshapen inflorescence with a shortened floral axis. Our results suggest that establishment of the complete C-function occurred gradually during AG lineage evolution even in gymnosperms.     

Over-Expression of <Emphasis Type="Italic">PmHSP17.9</Emphasis> in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Confers Thermotolerance

Small heat shock proteins (sHSPs) have been shown to be involved in stress tolerance. However, their functions in Prunus mume under heat treatment are poorly characterized. To improve our understanding of sHSPs, we cloned a sHSP gene, PmHSP17.9, from P. mume. Sequence alignment and phylogenetic analysis indicated that PmHSP17.9 was a member of plant cytosolic class III sHSPs. Besides heat stress, PmHSP17.9 was also upregulated by salt, dehydration, oxidative stresses and ABA treatment. Leaves of transgenic Arabidopsis thaliana that ectopically express PmHSP17.9 accumulated less O₂ ^? and H₂O₂ compared with wild type (WT) after 42 °C treatment for 6 h. Over-expression of PmHSP17.9 in transgenic Arabidopsis enhanced seedling thermotolerance by decreased relative electrolyte leakage and MDA content under heat stress treatment when compared to WT plants. In addition, the induced expression of HSP101, HSFA2, and delta 1-pyrroline-5-carboxylate synthase (P5CS) under heat stress was more pronounced in transgenic plants than in WT plants. These results support the positive role of PmHSP17.9 in response to heat stress treatment.