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.     

LcMKK,a MAPK kinase from <Emphasis Type="Italic">Lycium chinense</Emphasis>, confers cadmium tolerance in transgenic tobacco by transcriptional upregulation of ethylene responsive transcription factor gene

Cadmium (Cd) is a highly toxic element to plants. Ethylene is an important phytohormone in the regulation of plant growth, development and stress response. Mitogen-activated protein kinase (MAPK) activation has been observed in plants exposed to Cd stress and was suggested to be involved in ethylene biosynthesis. We hypothesized that there may be a link between MAPK cascades and ethylene signalling in Cd-stressed plants. To test this hypothesis, the expression of LcMKK, LchERF and LcGSH1 genes, endogenous ethylene accumulation, GSH content and Cd concentration in Lycium chinense with or without Cd stress treatment were studied. Our results showed that LcMKK gene expression can be induced by the treatment of Cd in L. chinense. The transgenic tobacco expressing 35S::LcMKK showed greater tolerance to Cd stress and enhanced expression of NtERF and NtGSH1 genes, indicating that LcMKK is associated with the enhanced expression level of ERF and GSH synthesis-related genes in tobacco. We also found that endogenous ethylene and GSH content can be induced by Cd stress in L. chinense, and inhibited by cotreatment with PD98059, an inhibitor of MAPK kinase. Evidences presented here suggest that under Cd stress, GSH accumulation occurred at least partially by enhanced LcMKK gene expression and the ethylene signal transduction pathways might be involved in this accumulation.     

Characterization of a maize ERF gene, <Emphasis Type="Italic">ZmERF1,</Emphasis> in hormone and stress responses

ERFs are downstream component in ethylene signaling pathway and involved in plant’s abiotic stress response. The specific role of ERFs under stress and the molecular mechanism underlying the signaling cross talk still need to be elucidated. This study describes the isolation and characterization of ZmERF1 promoter. There were many cis-regulatory elements related to stress responses in the ZmERF1 promoter sequence. ZmERF1 could be highly induced by ABA and ethylene treatment in maize, suggesting that it might be at the crossroads of multiple hormone signaling pathways. Furthermore, ZmERF1 transgenic Arabidopsis lines (35S::ZmERF1) showed higher salt-tolerant, drought- and heat resistance. Consistently, tolerance-related genes were up-regulated in 35S::ZmERF1 lines compared with the WT plants in Arabidopsis. Overall, ZmERF1 might play an important role in plant resistance to a coercive environment by mediating various physiological processes via ethylene and ABA signaling pathways.     

Interactions between the grass shrimp <Emphasis Type="Italic">Palaemonetes pugio</Emphasis> and the salt marsh grasses <Emphasis Type="Italic">Phragmites australis</Emphasis> and <Emphasis Type="Italic">Spartina alterniflora</Emphasis>

The grass shrimp Palaemonetes pugio, a species common to Spartina alterniflora-dominated marshes, may be sensitive to the invasion of the common reed Phragmites australis in northeastern US salt marshes. We examined two questions: (1) Do grass shrimp have a preference for the native plant over the non-native plant? (2) Are grass shrimp more effective foragers on P. australis? We tested the first hypothesis by comparing the amount of time shrimp spend in physical contact with the plant types over a 1-h period. Shrimp were observed under different arrangements of vegetation to control for differences in conspicuous structural features. Additionally, the amount of time shrimp spent foraging on S. alterniflora and P. australis shoots was compared to determine if shrimp graze more often on S. alterniflora. Shrimp spent significantly more time in contact with S. alterniflora only when plant types were grouped at opposite ends of aquaria, but did not exhibit a foraging preference for this plant type. To address our second question, we investigated the effects of shrimp foraging on stem epifauna, an assemblage of semi-aquatic invertebrates associated with macrophyte shoots. Previous research showed that P. australis supports a lower density of stem-dwelling epifauna relative to S. alterniflora. We hypothesized that the primary grazer of this community, P. pugio, can forage on P. australis stems more effectively due to structural differences between the two plants, causing the lower abundance of epifauna through top-down effects. We exposed individual shoots inhabited by epifauna to shrimp and compared faunal densities on exposed shoots to densities on control shoots after 18 h. The reduction of epifauna by predation was proportional on the two plant types. Therefore, top-down effects can be ruled out as an explanation for the patchy distribution of epifauna observed in P. australis–S. alterniflora marshes.     

Functional analysis of <Emphasis Type="Italic">PI-</Emphasis>like gene in relation to flower development from bamboo (<Emphasis Type="Italic">Bambusa oldhamii</Emphasis>)

Bamboo flowering owns many unique characteristics and remains a mystery. To investigate the molecular mechanisms underlying flower development in bamboo, a petal-identity gene was identified as a PISTILLATA homologue named BoPI from Bambusa oldhamii (bamboo family). Expression analysis showed that BoPI was highly expressed in flower organs and gradually increased during flower development stage, suggesting that BoPI played an important role in flower development. Ectopic expression of BoPI in Arabidopsis caused conversion of sepals to petals. 35S::BoPI fully rescued the defective petal formation in the pi-1 mutant. BoPI could interact with BoAP3 protein in vitro. These results suggested that BoPI regulated flower development of bamboo in a similar way with PI. Besides flower organs, BoPI was also expressed in leaf and branch, which revealed that BoPI may involve in leaf and branch development. Similar to other MIKC-type gene, BoPI contained the C-terminal sequence but its function was controversial. Ectopic expression of the C-terminal deletion construct (BoPI- ΔC) in Arabidopsis converted sepals to petals; BoPI- ΔC interacted with BoAP3 on yeast two-hybrid assay, just like the full-length construct. The result implied that the C-terminal sequence may not be absolutely required for organ identity function in the context of BoPI.     

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">CKB1</Emphasis> is involved in abscisic acid and gibberellic acid signaling to regulate stress responses in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Casein kinase II (CK2), an evolutionarily well-conserved Ser/Thr kinase, plays critical roles in all higher organisms including plants. CKB1 is a regulatory subunit beta of CK2. In this study, homozygous T-DNA mutants (ckb1-1 and ckb1-2) and over-expression plants (35S:CKB1-1, 35S:CKB1-2) of Arabidopsis thaliana were studied to understand the role of CKB1 in abiotic stress and gibberellic acid (GA) signaling. Histochemical staining showed that although CKB1 was expressed in all organs, it had a relatively higher expression in conducting tissues. The ckb1 mutants showed reduced sensitivity to abscisic acid (ABA) during seed germination and seedling growth. The increased stomatal aperture, leaf water loss and proline accumulation were observed in ckb1 mutants. In contrast, the ckb1 mutant had increased sensitivity to polyaluminum chloride during seed germination and hypocotyl elongation. We obtained opposite results in over-expression plants. The expression levels of a number of genes in the ABA and GA regulatory network had changed. This study demonstrates that CKB1 is an ABA signaling-related gene, which subsequently influences GA metabolism, and may play a positive role in ABA signaling.     

A nodule endophytic plant growth-promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> and its effects on growth,nodulation and metal uptake in <Emphasis Type="Italic">Medicago lupulina</Emphasis> under copper stress

The aim of this study was to determine the plant growth-promoting potential of the nodule endophytic Pseudomonas brassicacearum strain Zy-2-1 when used as a co-inoculant of Medicago lupulina with Sinorhizobium meliloti under copper (Cu) stress conditions. Strain Zy-2-1 was capable of producing ACC deaminase activity, IAA and siderophores, and was able to grow in the presence of Cu²⁺ up to 2.0 mmol/L. Co-inoculation of S. meliloti with Zy-2-1 enhanced M. lupulina root fresh weight, total plant dry weight, number of nodules, nodule fresh weight and nitrogen content in the presence of 100 or 300 mg/kg Cu²⁺. In the presence of 500 mg/kg Cu²⁺, co-inoculation with S. meliloti and strain Zy-2-1 increased plant height, number of nodules, nodule fresh weight and nitrogen content in comparison to S. meliloti inoculation alone. Furthermore, a higher amount of Cu accumulation in both shoots and roots and a higher level of Cu translocation to shoots were observed in co-inoculated plants. These results demonstrate that co-inoculation of M. lupulina with S. meliloti and P. brassicacearum Zy-2-1 improves plant growth, nitrogen nutrition and metal extraction potential. This can be of practical importance in the remediation of heavy metal-contaminated soils.     

Two orthologs of late blight resistance gene <Emphasis Type="Italic">R1</Emphasis> in wild and cultivated potato

The R1 gene for resistance to oomycete Phytophthora infestans (Mont.) de Bary, the causal agent of late blight disease of potato (Solanum tuberosum L.), was initially identified in S. demissum and potato varieties bred by introgressing the S. demissum germplasm. Later a sequence characterized amplified region (SCAR) marker R1-1205 of this gene was also found in S. stoloniferum and S. polytrichon. Here we describe the full-length R1 sequence cloned from S. stoloniferum. This sequence is translatable, and this evidence of structural gene integrity is reinforced by functional characterization of the S. stoloniferum R1 gene in an effectoromics experiment. When screened across a series of S. demissum and S. stoloniferum accessions, the R1 sequences differed by several single nucleotide polymorphisms and an indel; this indel served the basis for constructing SCAR markers R1-517 and R1-513 that reliably discerned two R1 orthologs. The demissum-specific marker R1-517 was found in all S. demissum accessions under study; it was also present in many demissum-derived potato varieties and hybrids. The stoloniferum-specific marker R1-513 was found in 27% of S. stoloniferum and S. polytrichon accessions; however, we failed to discern this marker in the genotypes of cultivated potato listing S. stoloniferum in their pedigrees. Most probably, such absence of R1-513 is best explained by an opportunistic breeding history of stoloniferum-derived founder lines, which were employed first and foremost in breeding for resistance to potato virus Y: eventually, these founder lines are devoid of the R1 gene.     

Molecular cloning and expression analysis of two <Emphasis Type="Italic">FAD2</Emphasis> genes from chia (<Emphasis Type="Italic">Salvia hispanica</Emphasis>)

FATTY ACID DESATURASE 2 (FAD2, EC 1.3.1.35), also known as delta-12 oleate desaturase, is a key enzyme for linoleic acid and α-linolenic acid biosynthesis. Chia (Salvia hispanica) seeds contain the highest known proportion of α-linolenic acid in any plant sources. In this study, two full-length FAD2 genes, named as ShFAD2-1 and ShFAD2-2, were isolated from S. hispanica based on RACE method. Both ShFAD2-1 and ShFAD2-2 proteins possess strong transmembrane helices, three histidine motifs and a C-terminal ER-located signal (YNNKL). Phylogenetic analysis showed that both ShFAD2-1 and ShFAD2-2 are grouped with constitutive plant FAD2s. Heterologous expression in Saccharomyces cerevisiae indicated that ShFAD2-1 and ShFAD2-2 genes both encode a bio-functional delta-12 oleate desaturase. ShFAD2-2 was mainly expressed in flowers and early-stage seeds while ShFAD2-1 expression was almost constitutive in different organs. qRT-PCR results demonstrated that ShFAD2-1 and ShFAD2-2 show a cold-induced and heat-repressed expression pattern, whereas they also were differentially up-regulated or repressed by other abiotic stresses. This is the first cloning and function characterization of FAD2 from S. hispanica, which can provide insights into molecular mechanism of high ALA traits of S. hispanica and enrich our understanding of the roles of FAD2 genes in various abiotic stresses.     

Genetic Control of the Wavy Shoots Character in Radish <Emphasis Type="Italic">Raphanus sativus</Emphasis> L.

The results of genetic analysis of the wavy shoots character supported the hypothesis on the existence of two genes, Wsh1 and Wsh2, whose mutations caused wavy shoots in radish lines 16 and 26, respectively. As shown by analysis of joint inheritance with marker characters, wavy shoots were inherited independently of the biochemical markers (genes Lap1, Ep, Dia1, and Aat). Gene Wsh1 was shown to be linked to genes A, ac, and ar; the mode of inheritance depended on the cross combination. In hybrids with mutant line 16, plants with novel morphological defects, which were absent in the parental forms, were detected. Possible mechanisms of new morphological abnormalities and change of linkage in the combinations analyzed are discussed.     

Phosphorylation regulates interaction of 210-kDa myosin light chain kinase <Emphasis Type="Italic">N</Emphasis>-terminal domain with actin cytoskeleton

High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303–314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

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.     

In vitro multiplication and genetic stability of two species of <Emphasis Type="Italic">Micranthocereus</Emphasis> Backeb. (Cactaceae) endemic to Bahia,Brazil

The two targeted species of this study, Micranthocereus flaviflorus subsp. densiflorus and M. polyanthus subsp. alvinii, are endemic to the state of Bahia and have ornamental value. The main goal of this work was to micropropagate these species and to evaluate the genetic stability of the regenerated plants. To do so, shoots originated from in vitro germinated plants were inoculated in MS/2 (Murashige, Skoog, Physiol Plant 15:473–497, 1962) media containing 1.34 μmol L^?1 of α-naphthaleneacetic acid (NAA) for morphogenesis induction. This was repeated for three consecutive subcultures, and the subcultured shoots were designated by order of production as S1, S2 and S3. Retention of morphogenic potential and acceleration of organogenic response was observed after the three subsequent propagation events, so that if shoots were used as explant source the in vitro propagation of M. flaviflorus could be achieved in 90 days and that of M. polyanthus could be optimized to 60 days of duration. In order to perform genetic stability analysis along subcultures, ISSR markers were used and genetic variation between shoots of each subculture and their donor plant was measured with Jaccard’s similarity coefficient. This analysis revealed high genetic stability in the in vitro propagation of all the donor plants of M. flaviflorus and M. polyanthus in regards to three consecutive shoot subcultures, in which similarities were 100% for both species. The study of a greater number of subcultures is suggested to assess morphogenesis potential and genetic fidelity in long term.