Co-Shine Wang

A Lily ASR Protein Involves Abscisic Acid Signaling and Confers Drought and Salt Resistance in Arabidopsis

LLA23, an abscisic acid-, stress-, and ripening-induced protein, was previously isolated from lily (Lilium longiflorum) pollen. The expression of LLA23 is induced under the application of abscisic acid (ABA), NaCl, or dehydration. To provide evidence on the biological role of LLA23 proteins against drought, we used an overexpression approach in Arabidopsis (Arabidopsis thaliana). Constitutive overexpression of LLA23 under the cauliflower mosaic virus 35S promoter confers reduced sensitivity to ABA in Arabidopsis seeds and, consequently, a reduced degree of seed dormancy. Transgenic 35S∷LLA23 seeds are able to germinate under unfavorable conditions, such as inhibitory concentrations of mannitol and NaCl. At the molecular level, altered expression of ABA/stress-regulated genes was observed. Thus, our results provide strong in vivo evidence that LLA23 mediates stress-responsive ABA signaling. In vegetative tissues, it is intriguing that Arabidopsis 35S∷LLA23 stomata remain opened upon drought, while transgenic plants have a decreased rate of water loss and exhibit enhanced drought and salt resistance. A dual function of the lily abscisic acid-, stress-, and ripening-induced protein molecule is discussed.

Two Classes of Proteins and mRNAs in Lilium longiflorum L. Identified by Human Vitronectin Probes

Vitronectin (VN) is a substrate adhesion molecule, an extracellular matrix glycoprotein that facilitates cell adhesion and cell movement in animals. We have reported the cross-reactivity of a 55-kD protein in plants with rabbit anti-human VN antibodies and the presence of VN-like sequences in plant genomes using a human VN cDNA probe. We have extended these studies by using human VN riboprobes to detect VN-like mRNAs in lily (Lilium longiflorum L.) and soybean. In both species, two mRNAs were detected. We have also identified a new cross-reactive protein (41 kD) using a different preparation of human VN antiserum. In lily roots five 41-kD isoforms were observed, whereas only three of these isoforms accumulated in leaves. Monospecific antibodies prepared against the plant proteins cross-reacted with the human VN protein and vice versa. We have purified the 41-kD protein using two-dimensional gel electrophoresis, and amino acid composition analysis indicates that it is similar in composition to human VN.

AtRH57, a DEAD-box RNA helicase, is involved in feedback inhibition of glucose-mediated abscisic acid accumulation during seedling development and additively affects pre-ribosomal RNA processing with high glucose.

The Arabidopsis thalianaT-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth.

Gene expression pattern at desiccation in the anther of Lilium longiflorum.

Although gene expression profile of pollen has been described, there is limited information regarding a particular phase during anther/pollen development. This work characterizes gene expression pattern at desiccation in lily (Lilium longiflorum Thunb. cv Snow Queen) anthers. We have applied a suppression-subtractive hybridization (SSH) strategy, through which 90 clones were identified and sequenced. These clones resulted in the identification of 42 individual cDNAs among which 33 genes were specifically expressed at the desiccation phase of anthers of >150-mm buds. Fourteen cDNAs were chosen for further examination. Six genes were both dehydration- and abscisic acid (ABA)-inducible whereas the other eight genes were apparently dehydration-irrelevant. The group of dehydration- and ABA-induced genes was also induced by desiccation that developmentally occurs in the anther. The application of fluridone has a significant effect of inhibition on mRNA accumulation of these genes in maturing anthers during which desiccation occurs. Pollen germination analysis indicated that, of those dehydration-irrelevant genes, three were ABA-responsive and the other five were not. Thus, three separate signal pathways that function in the activation of late genes at desiccation during anther development are established. The first is the ABA-dependent pathway induced by environmental stress of dehydration. The other two pathways of signaling triggered by developmental cues, through which one is ABA-dependent and another is ABA-independent. The 14 gene proteins showed spatial and temporal expression patterns and may participate in membrane/cell wall synthesis, cytoskeletal organization, signaling, RNA binding, ubiquitin-mediated degradation and transportation during germination and tube growth.

Rop GTPase and Its Target Cdc42/Rac-Interactive-Binding Motif-Containing Protein Genes Respond to Desiccation during Pollen Maturation

Here, we report unique desiccation-associated ABA signaling transduction through which the Rop (Rho GTPase of plants) gene is regulated during the stage of pollen maturation. A gene encoding Rho GTPase was identified in lily (Lilium longiflorum Thunb.) pollen. Phylogenetic tree analysis of lily LLP-Rop1 revealed that the protein shares greatest similarity with Group 4 Rops. The LLP-Rop1 gene was spatially and temporally regulated in lily plants during anther development. Accumulation of the LLP-Rop1 transcript decreased its level of accumulation while LLP-12-2, a Rop-interactive CRIB motif-containing (RIC) transcript increased either by premature drying of developing anther/pollen or by the exogenous application of various concentrations of abscisic acid (ABA) during pollen maturation and tube growth. Application of norflurazon, an ABA biosynthesis inhibitor, also resulted in the downregulation of the LLP-Rop1 gene while LLP-12-2 was upregulated by ABA. Furthermore, an increase in ABA in the maturing pollen correlated with desiccation that occurred in the anther prior to anthesis. LLP-Rop1 overexpression inhibited tube elongation, and caused tube expansion and the formation of a ballooned tip. CFP-LLP-Rop1 was localized to the cytoplasm having a greater intensity along the tube plasma membrane. Fluorescence resonance energy transfer analysis of lily pollen tubes coexpressing CFP-LLP-Rop1 and YFP-LLP-12-2 demonstrated that LLP-12-2 is a target RIC protein of active LLP-Rop1, but the interaction between LLP-Rop1 and LLP-12-2 proteins is probably irrelevant of dehydration in the dried pollen.

Expression and regulation of two novel anther-specific genes in Lilium longiflorum.

Two stage-specific genes have been isolated from a subtractive cDNA library constructed from developing anthers of lily (Lilium longiflorum). The proteins encoded by the two genes have a strong hydrophobic region at the N-terminus, indicating the presence of a signal peptide. The deduced LLA-67 is a new type of small cysteine-rich protein whose sequence exhibits four consecutive CX(3)CX(6-10) repeats that could form signal-receiving finger motifs, while the deduced LLA-115 protein shows significant similarities to a rice unknown protein, and putative cell wall proteins of Medicago truncatula and Arabidopsis. The transcripts of LLA-67 and LLA-115 were anther specific and differentially detected at the phase of microspore development. In situ hybridization with antisense riboprobes of the two genes in the anther showed strong signals localized to the tapetal layer of the anther wall. The LLA-67 mRNA was also detected in the microspore at the phase of microspore development but the LLA-115 mRNA was not. The LLA-115 gene can be exogenously induced by gibberellin (GA), whereas the LLA-67 gene cannot be induced. Studies with the GA biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that the two genes were negatively regulated by ethylene and a cross-talk between GA and ethylene was involved in the regulation of the two genes occurring in young anthers. The treatment of NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development to a status close to that of control. DNA blots of lily genomic DNA indicated that the two genes were encoded by a small gene family. The different actions of hormones on gene expression and the possible function of the gene products in young anthers are discussed.

The LLA23 protein translocates into nuclei shortly before desiccation in developing pollen grains and regulates gene expression in Arabidopsis

We have isolated the LLA23 gene in the pollen of Lilium longiflorum. The LLA23 gene encodes an ASR (named after abscisic acid, stress and ripening) protein that has a nuclear localization sequence at the C terminus. The gene is interrupted by one single intron and possesses a long 5′-untranslated region. Southern blots of lily genomic DNA indicated that LLA23 is a member of a small gene family. We examined the link between LLA23 location and the desiccation that naturally occurs in developing anthers using immunogold labeling. When pollen reached maturity, a significant increase in LLA23 labeling was observed in the nuclei of both vegetative and generative cells from 10- to 12-cm buds and thereafter. This clearly demonstrates that a marked increase in LLA23 translocation from the cytoplasm to both nuclei of pollen grains occurs in 12-cm buds, a stage shortly before the commencement of desiccation during anther development. In addition, microarray analysis showed that 410 (206 up-regulated and 204 down-regulated) genes have altered expression in LLA23-overexpressing plants. Quantitative PCR analysis confirmed the changes in mRNA levels observed in our microarray analysis. This genome-wide overview of gene expression supports the theory that LLA23 acts as a regulator.

New Insights into Desiccation-Associated Gene Regulation by Lilium longiflorum ASR during Pollen Maturation and in Transgenic Arabidopsis

LLA23, a member of the abscisic acid-, stress-, and ripening-induced (ASR) protein family, was previously isolated from lily (Lilium longiflorum) pollen. The lily ASR is induced through desiccation-associated ABA signaling transduction in the pollen. ASRs are highly hydrophilic and intrinsically unstructured proteins with molecular masses generally less than 18 kDa. LLA23 is abundant in the cytoplasm and nuclei of both vegetative and generative cells of pollen grains. The protein in the nucleus and in the cytoplasm is partly regulated by dehydration. A dual role is proposed for LLA23, as a regulator and a protective molecule, upon exposure to water deficits. This chapter reviews the current state of literature on Asr genes, protein structure, function, and their responses to various stresses. In a study, a genome-wide microarray was used to monitor the expression of LLA23-regulated genes, focusing on the relationship between ASR-, glucose-, and drought-inducible genes, and outlined the difference and cross talk of gene expression among these signaling networks. A strong association was observed in the expression of stress-responsive genes and found 25 genes that respond to all three treatments. Highly inducible genes were also found in each specific stress treatment. Promoter sequence analysis of LLA23-inducible genes enabled us not only to identify possible known cis-acting elements in the promoter regions but also to expect the existence of novel cis-acting elements involved in ASR-responsive gene expression. ASR can be used to improve crops and economically important plants against various environmental stresses.

Identification of the tapetum/microspore-specific promoter of the pathogenesis-related 10 gene and its regulation in the anther of Lilium longiflorum.

A tapetum/microspore-specific pathogenesis-related (PR) 10 gene was previously identified in lily (Lilium longiflorum Thunb.) anthers. In situ hybridization and RNA blot analysis indicated that the lily PR10 genes are expressed specifically and differentially in the tapetum of the anther wall and in microspores during anther development. The accumulation of PR10 transcripts was exogenously induced by gibberellic acid (GA) and was suppressed by ethylene. Studies using inhibitors of GA and ethylene revealed that the lily PR10 is modulated by an antagonistic interaction between GA and ethylene. The treatment of norbornadien, an ethylene inhibitor, caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole, an inhibitor of GA biosynthesis, arrested tapetal development to a status close to that of control. The expression of the lily PR10g promoter in transgenic Arabidopsis was determined using the β-glucuronidase (GUS) reporter gene indicated that the decisive fragment required for anther specificity is located -1183 bp to -880 bp upstream of the transcription start site. The PR10gPro::barnase transgenic lines exhibited complete male sterility because of the disruption of the tapetum and the deformation of microspore/pollen. The anther specificity of lily PR10 highlights the importance of the tapetum/microspore-specific PR10g promoter for future biotechnological and agricultural applications.

A desiccation-induced transcript in lily (Lilium longiflorum) pollen

Summary This work characterizes a desiccation and abscisic acid (ABA)-induced transcript, designated LLP-B3, present in pollen of Lilium longiflorum during development and stress. The LLP-B3 cDNA encodes a gene product having a sequence of 160 amino acids, a calculated molecular mass of 17.8 kDa, and a pI of 4.3. The polypeptide is mainly hydrophilic having a stretch of hydrophobic region at the N-terminus, indicating the presence of a signal peptide. The expression of LLP-B3 gene is pollen-specific, and the transcript accumulates only at the stage of pollen maturation. Premature drying of developing pollen indicated that the accumulation of LLP-B3 transcripts was associated with desiccation. Treatment of pollen with abscisic acid resulted in the accumulation of LLP-B3 mRNA during germination. Sequence analysis demonstrates similarities between the predicted lily LLP-B3 protein and a corn Zmc13, a tomato LAT52, an arabidopsis leaf SAH7 and a group of major allergens including Ole e 1 of olive tree. Southern blots of lily genomic DNA indicate that LLP-B3 is a single or low copy number gene. The function of the desiccation-associated protein is discussed.