Chin-Ying Yang

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.

The AP2/ERF transcription factor AtERF73/HRE1 modulates ethylene responses during hypoxia in Arabidopsis

A number of APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) genes have been shown to function in abiotic and biotic stress responses, and these genes are often induced by multiple stresses. We report here the characterization of an AP2/ERF gene in Arabidopsis (Arabidopsis thaliana) that is specifically induced during hypoxia. We show that under normoxic conditions, the expression of AtERF73/HRE1 can be induced by exogenous addition of 1-aminocyclopropane-1-carboxylic acid and that a combination of hypoxia and 1-aminocyclopropane-1-carboxylic acid results in hyperinduction of AtERF73/HRE1 expression. In addition, hypoxic induction of AtERF73/HRE1 is reduced but not completely abolished in ethylene-insensitive mutants and in the presence of inhibitors of ethylene biosynthesis and responses. These results suggest that, in addition to ethylene, an ethylene-independent signal is also required to mediate hypoxic induction of AtERF73/HRE1. To assess the role of AtERF73/HRE1, we generated three independent RNA interference (RNAi) knockdown lines of AtERF73/HRE1. Under normoxic conditions, the AtERF73/HRE1-RNAi seedlings displayed increased ethylene sensitivity and exaggerated triple responses, indicating that AtERF73/HRE1 might play a negative regulatory role in modulating ethylene responses. Gas chromatography analyses showed that the production of ethylene was similar between wild-type and RNAi lines under hypoxia. Quantitative reverse transcription-polymerase chain reaction analyses showed that hypoxia-inducible genes could be affected by AtERF73/HRE1-RNAi lines in two different ways: hypoxic induction of glycolytic and fermentative genes was reduced, whereas induction of a number of peroxidase and cytochrome P450 genes was increased. Taken together, our results show that AtERF73/HRE1 is involved in modulating ethylene responses under both normoxia and hypoxia.

Hydrogen peroxide controls transcriptional responses of ERF73/HRE1 and ADH1 via modulation of ethylene signaling during hypoxic stress

Hypoxia, or oxygen deficiency, is an abiotic stress that plants are subjected to during soil flooding. Therefore, plants have evolved adaptive mechanisms to sense oxygen deficiency and make coordinated changes at the transcriptional level. The results of this study show that the interplay between hydrogen peroxide and ethylene affected the transcriptional responses of ERF73/HRE1 and ADH1 during hypoxia signaling. H2O2 affected the abundance of ERF73/HRE1 and ADH1 mRNAs in both wild-type Arabidopsis and the ethylene-insensitive mutant, ein2-5. Promoter analysis was conducted using transgenic plants expressing an ERF73/HRE1 promoter–β-glucuronidase reporter gene construct. GUS staining observations and activity assays showed that GUS was regulated similarly to, and showed a similar accumulation pattern as, H2O2 during hypoxia. The transcript levels of ERF73/HRE1 and ADH1 were significantly decreased in the WT by combined hypoxia and diphenylene iodonium chloride (DPI, an NADPH oxidase inhibitor) treatment. In ein2-5, induction of ERF73/HRE1 was also reduced significantly by the combined hypoxia and DPI treatment. In contrast, ADH1 mRNA levels only slightly decreased after this treatment. When DPI was supplied at different time points during hypoxia treatment, H2O2 had critical effects on regulating the transcript levels of ERF73/HRE1 and ADH1 during the early stages of hypoxia signaling. The induction of hypoxia-inducible genes encoding peroxidases and cytochrome P450s was affected, and accumulation of H2O2 was reduced, in ein2-5 during hypoxic stress. Together, these results demonstrate that H2O2 plays an important role during primary hypoxia signaling to control the transcriptional responses of ERF73/HRE1 and ADH1 via modulation of ethylene signaling.

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.

Ethylene and hydrogen peroxide are involved in hypoxia signaling that modulates AtERF73/HRE1 expression

Reactive oxygen species (ROS) have been reported to trigger signaling pathways that interact with other signaling pathways mediated by nitric oxide, lipid messengers, and plant hormones. In a previous study, we demonstrated that ethylene was involved in hypoxia signaling to regulate the expression of downstream genes such as AtERF73/HRE1 and ADH1. Furthermore, H2O2 and ethylene interplay has an effect on AtERF73/HRE1 and ADH1 expression during the early stages of hypoxia signaling. Here, we propose a model for the main transcription factor AtERF73/HRE1, which is controlled by 3 pathways during hypoxia. These include an ethylene-dependent pathway, an ethylene-independent/H2O2-dependent pathway, and an ethylene and H2O2-independent pathway involved in hypoxia signaling to modulate AtERF73/HRE1.

Physiological Responses and Expression Profile of NADPH Oxidase in Rice (Oryza Sativa) Seedlings under Different Levels of Submergence.

BackgroundFlooding due to global climate change is a serious problem that frequently decreases crop yields. Rice fields in flood-prone areas often experience full or partial submergence. Submergence has an adverse effect on internal oxygen availability, sugar status and survival. Complete submergence imposes severe pressure on plants, principally because the excess water in their surroundings deprives them of certain basic resources such as oxygen, carbon dioxide and light for photosynthesis. To better understand the mechanisms involved under different levels of flooding, it is necessary to further observe physiological responses and to identify the Rboh genes involved and determine how they are regulated during submergence.ResultsIn this study, significant physiological changes were observed in plant height, leaf sheath elongation and chlorophyll a, b and total content under partial and full submergence treatments. Senescence-regulating genes were severely affected under full submergence. Additionally, intracellular oxidative homeostasis was disrupted by overproduction of H2O2 and O2−, which affected cell viability and antioxidant enzyme activity, under different levels of submergence. Quantitative RT-PCR analyses revealed that complex regulation of Rboh genes is involved under different levels of submergence.ConclusionOur results demonstrated that the effect of physiological and the transcript levels of OsRboh genes were presented different responses to different levels of submergence in rice seedlings. There have different mechanism in intracellular to response different levels of submergence. Finally we discuss effects of the regulation of OsRboh expression and ROS production which was important to maintain homeostasis to help rice seedlings face different levels of submergence.

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.

AtRBOH I confers submergence tolerance and is involved in auxin-mediated signaling pathways under hypoxic stress

Plants suffer from oxygen deficiency (hypoxia) and energy starvation under flooding conditions. Higher plants have evolved complex adaptive mechanisms to flooding that are induced by changes in the cellular redox state and phytohormones. Previously, we showed that the transcript levels of respiratory burst oxidase homolog I (AtRBOH I) in Arabidopsis increase under hypoxic stress. In this study, we used two independent Atrboh I-knockout lines to assess the molecular function of AtRBOH I in hypoxic signaling pathways. Under submergence conditions, the Atrboh I-knockout lines had a reduced survival rate and lower chlorophyll contents than those of wild type. The patterns of AtRBOH I expression were analyzed by fusing its promoter to the GUS reporter. These expression analyses indicated that AtRBOH I expression was activated by hypoxia, but this induction was reduced by the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Quantitative RT-PCR analyses showed that the transcript levels of hypoxia-inducible genes (AtHRE1, AtADH1, AtLDH, and AtSUS1) were reduced in AtRBOH I-knockout lines under hypoxic conditions. The transcript levels of AtSUS1 were lower in AtRBOH I-knockout lines than in wild type in the hypoxia combined with NPA treatment. Hypoxic conditions increased the transcript levels of the auxin-responsive genes At1g19840, At3g23030, and At5g19140, and hypoxia combined with NPA resulted in increased transcript levels of the ethylene biosynthetic genes AtACS7 and AtACS8. Together, these results show that AtRBOH I regulates the expression of genes involved in ethylene biosynthesis and down-stream of hypoxia signaling, and that there is some interplay between hypoxia signaling and auxin-mediated signaling pathways under hypoxic stress.

Molecular identification and characterization of a serine carboxypeptidase-like gene associated with abiotic stress in tea plant, Camellia sinensis (L.)

Tea (Camellia sinensis L.) contains secondary metabolites including polyphenolic proanthocyanidins (PAs) and their precursors. The SCPL (serine carboxypeptidase-like) proteins are a group of acyltransferase enzymes that modify plant natural products. We isolated CsSCPL, which encodes a SCPL protein, from oolong tea. Sequence alignment analyses showed that CsSCPL is a serine carboxypeptidase with high homology to other SCPLs, including those in persimmon, grape, woodland strawberry, and sweet orange. Quantitative RT-PCR analyses revealed that the highest transcript levels of CsSCPL were in young leaves of tea seedlings and buds of mature plants. CsSCPL transcription increased in response to heat but decreased in response to cold, high salinity, and drought. The degree of epigallocatechin galloylation increased after heat treatment and the degree of epicatechin galloylation decreased after cold treatment. In field-grown tea plants, the highest transcript levels of CsSCPL were in summer. Together, these results show that CsSCPL transcripts accumulate in one-tip-two-leaf tissues of oolong tea plants during the hottest parts of the growing season, and in response to abiotic stress. The degree of catechin galloylation was positively correlated with CsSCPL transcript levels after heat or cold treatments. Our results will be useful for further research on the functions of SCPLs in plants.