Isolation and Expression Analysis of <Emphasis Type="Italic">PeDWF1</Emphasis> in <Emphasis Type="Italic">Phyllostachys edulis</Emphasis>

Brassinosteroids (BRs) are steroidal hormones that play crucial roles in various processes of plant growth and development. DWF1 encodes a delta(24)-sterol reductase that participates in one of the early stage in the brassinosteroids’ biosynthetic pathway: the conversion of 24-methylenecholesterol to campesterol. Here we report the isolation and expression of one DWF1 homologous gene, PeDWF1, in moso bamboo (Phyllostachys edulis (Carrière) J. Houz.). Sequence analysis revealed that the open reading frame of PeDWF1 was 1686-bp encoding a protein composed of 561 amino acid residues with a calculated molecular weight of 65.1 kD and a theoretic isoelectric point of 8.32. Phylogenetic analysis indicated that PeDWF1 was very close to the cell elongation protein Dwarf1 in rice (Oryza sativa). Furthermore, transient expression of a PeDWF1::GFP fusion protein showed that PeDWF1 was an integral membrane protein most probably associated with the endoplasmic reticulum similar to Dwarf1. Tissue specific expression analysis showed that PeDWF1 was constitutively expressed in moso bamboo with the highest level in shoots and the lowest level in mature leaves. In the early growing stage of shoots, the expression level of PeDWF1 had a rising trend with the increasing height of shoots. These results indicated that PeDWF1 might be involved in the regulation of shoot development by participating in BRs biosynthesis. Moreover, PeDWF1 was heterologously expressed in Escherichia coli and the recombinant protein was about 65 kD, which facilitated further study on the gene function of PeDWF1 in bamboo.     

<Emphasis Type="Italic">PAD4</Emphasis>, <Emphasis Type="Italic">LSD1</Emphasis> and <Emphasis Type="Italic">EDS1</Emphasis> regulate drought tolerance,plant biomass production,and cell wall properties

Key message

Arabidopsis and poplar with modified PAD4, LSD1 and EDS1 genes exhibit successful growth under drought stress. The acclimatory strategies depend on cell division/cell death control and altered cell wall composition.

Abstract

The increase of plant tolerance towards environmental stresses would open much opportunity for successful plant cultivation in these areas that were previously considered as ineligible, e.g. in areas with poor irrigation. In this study, we performed functional analysis of proteins encoded by PHYTOALEXIN DEFICIENT 4 (PAD4), LESION SIMULATING DISEASE 1 (LSD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes to explain their role in drought tolerance and biomass production in two different species: Arabidopsis thaliana and Populus tremula × tremuloides. Arabidopsis mutants pad4-5, lsd1-1, eds1-1 and transgenic poplar lines PAD4-RNAi, LSD1-RNAi and ESD1-RNAi were examined in terms of different morphological and physiological parameters. Our experiments proved that Arabidopsis PAD4, LSD1 and EDS1 play an important role in survival under drought stress and regulate plant vegetative and generative growth. Biomass production and acclimatory strategies in poplar were also orchestrated via a genetic system of PAD4 and LSD1 which balanced the cell division and cell death processes. Furthermore, improved rate of cell division/cell differentiation and altered physical properties of poplar wood were the outcome of PAD4- and LSD1-dependent changes in cell wall structure and composition. Our results demonstrate that PAD4, LSD1 and EDS1 constitute a molecular hub, which integrates plant responses to water stress, vegetative biomass production and generative development. The applicable goal of our research was to generate transgenic plants with regulatory mechanism that perceives stress signals to optimize plant growth and biomass production in semi-stress field conditions.

     

Morphological and physiological characteristics of transgenic tobacco plants expressing expansin genes: <Emphasis Type="Italic">AtEXP10</Emphasis> from <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">PnEXPA1</Emphasis> from poplar

Expansins are non-enzymatic plant proteins breaking hydrogen bonds between cellulose microfibrils and hemicellulose polymer matrix. Each plant has many expansin genes, whose protein products participate in the regulation of plant growth and development mainly by regulating cell expansion. To analyze the effects of elevated expansin expression on the plant organ sizes, we cloned the AtEXPA10 gene from Arabidopsis thaliana and PnEXPA1 gene from Populus nigra. Transgenic tobacco plants expressing the target genes were obtained. The obtained transgenic tobacco plants were shown to have significantly larger leaves and longer stems compared to control plants. The flowers were quite insignificantly larger, but at the same time transgenic plants had more flowers. The microscopic studies showed that the organs of AtEXPA10-carrying plants were larger mainly due to stimulated cell proliferation, whereas the overexpression of the PnEXPA1 gene activated cell expansion.     

Accumulation and detoxification of cadmium in <Emphasis Type="Italic">Brassica pekinensis</Emphasis> and <Emphasis Type="Italic">B. chinensis</Emphasis>

The effect of excessive Cd on the growth and metal uptake by leafy vegetables Brassica chinensis L. (cv. Wuyueman) and Brassica pekinensis (Lour.) Rupr. (cv. Qingyan 87-114) were studied in hydroponic solution culture. The Cd concentration higher than 10 μM significantly decreased the root elongation and leaf chlorophyll contents of both plant species. The shoots of B. pekinensis had significantly higher concentrations of total and water-soluble Cd than B. chinensis. The roots of both species accumulated more Cd than the shoots in all the Cd treatments. Most of the Cd in the roots was found in the cell walls. The shoot/root ratio of Cd concentrations in B. pekinensis was always greater than that in B. chinensis. But, the concentration and proportion of Cd in the cell walls in B. chinensis were higher than that in B. pekinensis. Cadmium treatments also increased the concentrations of total non-protein thiols (NPT) in the shoots of the both species. A significant correlation was found between the concentrations of soluble Cd and NPT in plant shoots.     

<Emphasis Type="Italic">smyd1</Emphasis> and <Emphasis Type="Italic">smyd2</Emphasis> are expressed in muscle tissue in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Epigenetic modifications of histone play important roles for regulation of cell activity, such as cell division, cell death, and cell differentiation. A SET domain consisting of about 130 amino acids has lysine methyltransferase activity in the presence of the cosubstrate S-adenosyl-methionine. More than 60 SET domain-containing proteins have been predicted in various organisms. One of them, the SMYD family genes which contain a SET domain and a zinc-finger MYND domain are reported to regulate cell cycle and muscle formation. Here we examined the expression and function of smyd1 and 2 in Xenopus. smyd1 and 2 were expressed in various muscle tissues. While smyd1 expression was observed mainly in cardiac muscle and skeletal muscle, smyd2 expression was done abundantly in skeletal muscle and face region. Moreover, by loss-of-function experiments using antisense morpholino oligonucleotides, it was suggested that smyd1 and 2 related to muscle cells differentiation.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

Host physiological condition regulates parasitic plant performance: <Emphasis Type="Italic">Arceuthobium vaginatum</Emphasis> subsp. <Emphasis Type="Italic">cryptopodum</Emphasis> on <Emphasis Type="Italic">Pinus ponderosa</Emphasis>

Much research has focused on effects of plant parasites on host-plant physiology and growth, but little is known about effects of host physiological condition on parasite growth. Using the parasitic dwarf mistletoe Arceuthobium vaginatum subsp. cryptopodum (Viscaceae) and its host Pinus ponderosa, we investigated whether changes in host physiological condition influenced mistletoe shoot development in northern Arizona forests. We conducted two studies in two consecutive years and used forest thinning (i.e., competitive release) to manipulate host physiological condition. We removed dwarf mistletoe shoots in April, before the onset of the growing season, and measured the amount of regrowth in the first season after forest thinning (Study I: n=38 trees; Study II: n=35 trees). Thinning increased tree uptake of water and carbon in both studies, but had no effect on leaf N concentration or δ¹³C. Mistletoe shoot growth was greater on trees with high uptake of water and carbon in thinned stands than trees with low uptake in unthinned stands. These findings show that increased resource uptake by host trees increases resources to these heterotrophic dwarf mistletoes, and links mistletoe performance to changes in host physiological condition.     

Two active bamboo <Emphasis Type="Italic">mariner</Emphasis>-like transposable elements (<Emphasis Type="Italic">Ppmar1</Emphasis> and <Emphasis Type="Italic">Ppmar2</Emphasis>) identified as the transposon-based genetic tools for mutagenesis

Bamboo is one of the most important non-timber forest species in the world, but their molecular breeding lags far behind in contrast to other economic plants. Regarding the difficulties of hybridization and gene modification, the transposon-based insertional mutagenesis might be an alternative, feasible way for molecular breeding of bamboo. A systematic search for potential active transposons identified two full-length mariner-like elements (MLEs) (Ppmar1 and Ppmar2) from moso bamboo in the previous study. Both MLEs contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase. Two transposases contain intact DNA-binding motifs and a DD39D catalytic domain which indicates that Ppmar1 and Ppmar2 are likely active. Here, we deployed a heterologous transposition system of Arabidopsis thaliana to study the transposition activity of Ppmar1 and Ppmar2. The results show that both MLEs could transpose in A. thaliana. Excisions of Ppmar1 and Ppmar2 are usually unperfect as they leave 1–4 bp in excision sites. The reinsertions of both Ppmar1 and Ppmar2 occur at TA dinucleotides and prefer to insert into the TA-rich regions. The insertion sites are dispersed and non-linked. Two active bamboo transposons identified here not only could be applied to construction of the bamboo mutant libraries but also would provide another choice for other plant transposon-based gene tagging.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the dwarf pomegranate (<Emphasis Type="Italic">Punica granatum</Emphasis> L. var. <Emphasis Type="Italic">nana</Emphasis>)

The dwarf pomegranate (Punica granatum L. var. nana) is a dwarf ornamental plant that has the potential to be the model plant of perennial fruit trees because it bears fruits within 1 year of seedling. We established an Agrobacterium-mediated transformation system for the dwarf pomegranate. Adventitious shoots regenerated from leaf segments were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBin19-sgfp, which contains neomycin phosphotransferase (npt II) and green fluorescent protein (gfp) gene as a selectable and visual marker, respectively. After co-cultivation, the inoculated adventitious shoots were cut into small pieces to induce regeneration, and then selected on MS medium supplemented with 0.5 μM α-naphthaleneacetic acid (NAA), 5 μM N⁶-benzyladenine (BA), 0.3% gellan gum, 50 mg/l kanamycin, and 10 mg/l meropenem. Putative transformed shoots were regenerated after 6–8 months of selection. PCR and PCR-Southern blot analysis revealed the integration of the transgene into the plant genome. Transformants bloomed and bore fruits within 3 months of being potted, and the inheritance of the transgene was confirmed in T₁ generations. The advantage of the transformation of dwarf pomegranate was shown to be the high transformation rate. The establishment of this transformation system is invaluable for investigating fruit-tree-specific phenomena.     

<Emphasis Type="Italic">AtTBP2</Emphasis> and <Emphasis Type="Italic">AtTRP2</Emphasis> in <Emphasis Type="Italic">Arabidopsis</Emphasis> encode proteins that bind plant telomeric DNA and induce DNA bending in vitro

Telomeric DNA-binding proteins (TBPs) are crucial components that regulate the structure and function of eukaryotic telomeres and are evolutionarily conserved. We have identified two homologues of AtTBP1 (for Arabidopsis thaliana telomeric DNA binding protein 1), designated as AtTBP2 and AtTRP2, which encode proteins that specifically bind to the telomeric DNA of this plant. These proteins show extensive homology with other known plant TBPs. The isolated C-terminal segments of these proteins were capable of sequence-specific binding to duplex telomeric plant DNA in vitro. DNA bending assays using the Arabidopsis TBPs revealed that AtTBP1 and AtTBP2 have DNA-bending abilities comparable to that of the human homologue hTRF1, and higher than those of AtTRP1 and AtTRP2.     

Thidiazuron Promotes <Emphasis Type="Italic">In Vitro</Emphasis> Plant Regeneration of <Emphasis Type="Italic">Arachis correntina</Emphasis> (<Emphasis Type="Italic">Leguminosae</Emphasis>) via Organogenesis

Arachis correntina (Burkart) Krapov. & W.C. Gregory is a herbaceous perennial leguminous plant growing in the Northeast of the Province Corrientes, Argentina. It is important as forage. The development of new A. correntina cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the plant regeneration potential of mature leaves of A. correntina in tissue culture. Buds were induced from both petiole and laminae on 0.7% agar-solidified medium containing 3% sucrose, salts and vitamins from Murashige and Skoog (MS) supplemented with 0.5–25 M thidiazuron (TDZ). Shoot induction was achieved by transference of calli with buds to MS supplemented with 5 M TDZ. Fifty-four percent of the regenerated shoot rooted on MS + 5 M naphthaleneacetic acid. Histological studies revealed that shoots regenerated via organogenesis.     

Genomewide analysis of <Emphasis Type="Italic">ABCB</Emphasis>s with a focus on <Emphasis Type="Italic">ABCB1</Emphasis> and <Emphasis Type="Italic">ABCB19</Emphasis> in <Emphasis Type="Italic">Malus domestica</Emphasis>

The B subfamily of ATP-binding cassette (ABC) proteins (ABCB) plays a vital role in auxin efflux. However, no systematic study has been done in apple. In this study, we performed genomewide identification and expression analyses of the ABCB family in Malus domestica for the first time. We identified a total of 25 apple ABCBs that were divided into three clusters based on the phylogenetic analysis. Most ABCBs within the same cluster demonstrated a similar exon–intron organization. Additionally, the digital expression profiles of ABCB genes shed light on their functional divergence. ABCB1 and ABCB19 are two well-studied auxin efflux carrier genes, and we found that their expression levels are higher in young shoots of M106 than in young shoots of M9. Since young shoots are the main source of auxin synthesis and auxin efflux involves in tree height control. This suggests that ABCB1 and ABCB19 may also take a part in the auxin efflux and tree height control in apple.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of <Emphasis Type="Italic">Alnus acuminata</Emphasis> H.B.K. ssp. <Emphasis Type="Italic">acuminata</Emphasis> and its root nodulation by <Emphasis Type="Italic">Frankia</Emphasis>

A procedure for in vitro plant regeneration of Alnus acuminata from epicotyls with cotyledonary buds was developed using different media formulations with different growth regulators and carbon sources. The development of multiple buds on explants at the initiation step was obtained with MS at 1/2 strength with either 1 or 2M of BAP but not without it. Multiplication gave up to 15 elongating shoots by explant, the best medium being MS supplemented with vitamins from B5 medium, 1M of BAP and 87mM sucrose. Rooting of about 88% occurred in the medium MS with 83 mM sucrose and 1M IBA. Alnus acuminata did not developed well on WPM. Roots of in vitro propagated plants were nodulated by Alnus-infective Frankia. The root nodules show a typical alder root nodule anatomy and differentiation pattern and effectively fixed nitrogen. Rhamnaceae-infective Frankia did not nodulate in vitro cultivated Alnus acuminata suggesting that symbiotic recognition was not altered by in vitro regeneration of the plant.