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DNA Research Advance Access originally published online on April 21, 2006
DNA Research 2006 13(2):53-63; doi:10.1093/dnares/dsl001
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© The Author 2006. Kazusa DNA Research Institute
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

The Rice 14-3-3 Gene Family and its Involvement in Responses to Biotic and Abiotic Stress

Fang Chen, Qun Li, Liangxian Sun and Zuhua He*

National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences 300 Fenglin Road, Shanghai 200032, China

Received 19 September 2005; revised 22 March 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussions
 Acknowledgements
 References
 
14-3-3 proteins function as major regulators of primary metabolism and cellular signal transduction in plants. However, their involvement in plant defense and stress responses is largely unknown. In order to better address functions of the rice 14-3-3/GF14 proteins in defense and abiotic stress responses, we examined the rice GF14 family that comprises eight numbers. The phylogenetic comparison with the Arabidopsis 14-3-3 family revealed that the majority of rice GF14s might have evolved as an independent branch. At least four rice GF14 genes, GF14b, GF14c, GF14e and Gf14f were differentially regulated in the interactions of rice-Magnaporthe grisea and rice-Xanthomonas oryzae pv. oryzae, and the incompatible interactions stronger induced the genes than the compatible interactions. These GF14 genes were also induced by the defense compounds, benzothiadiazole, methyl jasmonate, ethephon and hydrogen peroxide. Similarly, they were differentially regulated by salinity, drought, wounding and abscisic acid. Tissue-specific analysis and expression of GF14-YFP fusions revealed that the four GF14 isoforms were expressed with tissue specificity and accumulated differentially in the cytoplasm and nucleus. Our current study provides fundamental information for the further investigation of the rice GF14 proteins.

Key words: rice; 14-3-3 family; biotic and abiotic stress; expression regulation; subcellular localization


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussions
 Acknowledgements
 References
 
All plant biological processes are controlled by signal transduction and metabolism regulation that have been known to occur via phosphorylation-mediated transition of protein states. In many cases, to complete their regulatory actions, these phosphorylated proteins must physically associate with the specialized adapter proteins, which are known as 14-3-3 phosphoserine/threonine binding proteins.1Go–4Go It is one hallmark of signal transduction and metabolism events to identify 14-3-3 participation in cellular regulatory pathways in which 14-3-3s, generally acting as adapters, chaperones, activators or repressors, interact physically with target (client) proteins phosphorylated to execute an important step in signal transduction and metabolism.

There are several lines of evidence to show that 14-3-3s are involved in many metabolic and signaling pathways for plant growth regulation and responses to environmental stress. The most prominent paradigms involving 14-3-3s in plant regulatory events include the regulation of plasma membrane H+-ATPase, nitrate reductase (NR) and sucrose phosphate synthase (SPS).1Go,4Go 14-3-3s have been known as positive regulators of H+-ATPase activity, with 14-3-3s and Mg2+ bound to the C-terminal region of the H+-ATPase, pump activity is stimulated. Interestingly, a wilt-inducing phytotoxin fusicoccin (FC) binds to the complex of 14-3-3s and H+-ATPase, resulting in continuous high-activity state of H+-ATPase, guard cell solute uptake and excessive transpiration that leads to leaf wilt.5Go–7Go Whereas, 14-3-3s function as inhibitors of NR and SPS. Mg2+ binding causes a structural change in 14-3-3s that allows binding to the phosphorylated target enzymes, a process known to inactivate NR and SPS.1Go 14-3-3 proteins also bind a range of transcription factors for their activation and translocation.8Go For example, the tobacco transcription factor RSG (repression of shoot growth) is bound to 14-3-3s and involved in GA biosynthesis.9Go,10Go

Interestingly, 14-3-3 proteins function as regulators of a wide range of target proteins that are involved in responses to abiotic and biotic stress, by regulating target proteins with functions of either signaling or transcription activation or defense.11Go For example, the tobacco T14-3-3 is induced in the perception of the salt stress12Go; two Arabidopsis 14-3-3 genes, RCI1/RCI1A and RCI2/RCI1B, are regulated during cold acclimation13Go; Arabidopsis 14-3-3 isoforms could bind to and probably activate a stress-responsive calcium-dependent protein kinase, CPK-1.14Go,15Go The activity of mitochondrial and chloroplast F0F1 ATP synthases, regulated by 14-3-3s, is involved in responses to light/dark transitions, anoxia changes in nutrient supply.16Go Recently, Yan et al.17Go reported that the transgenic cotton plants with constitutive expression of GF14{lambda} displayed phenotypes of ‘stay-green’ and enhanced tolerance to drought stress, probably through the regulation of the stomatal aperture by the complex of 14-3-3s and H+-ATPase. 14-3-3s have been found involved in plant defense responses.11Go,18Go For instance, a 14-3-3 gene was identified to be regulated in the non-host hypersensitive response (HR) between barley and Blumeria graminis f.sp. tritici.19Go Furthermore, it is well characterized that 14-3-3s bind to and activate H+-ATPase, creating a binding site for the phytotoxin FC, and FC-binding activity of an epidermal microsomal fraction increases upon the pathogen attack, suggesting that 14-3-3s are involved in an epidermis-specific response to the fungus probably through activating the proton pump (H+-ATPase) to stimulate the HR.20Go Caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT1) and ascorbate peroxidase implicated in plant defense or oxidative stress are identified to interact with 14-3-3s by yeast two-hybrid screening.21Go,22Go It is also shown that the white spruce and hybrid poplar 14-3-3s are up-regulated by wounding or wounding elicitors, or by chitosan and jasmonic acid, two defensive elicitors, and H+-ATPase is a potential target for the 14-3-3-mediated regulation during stress.23Go Similar to FC, other two inducers of programmed cell death, tunicamycin and brefeldin A, also induce the accumulation of 14-3-3 proteins.24Go Interestingly, a 14-3-3-interactor, AKR2, is an ankyrin-repeat containing protein and negatively regulates transcription factors that mediate defense responses.25Go,26Go The AKR2-antisense plants developed HR-like lesions with increased H2O2 generation and exhibited increased resistance to a bacterial pathogen.26Go

Plants have large 14-3-3 gene families, and various 14-3-3 isoforms have differential affinities for certain target proteins. The dicotyledonous model plant Arabidopsis has hitherto the most complete and largest 14-3-3 family which consists of 15 members with at least 12 expressed members classified into two evolutionary branches, the {varepsilon} group and the non-{varepsilon} group.3Go,4Go These 14-3-3 isoforms exhibit a high cell and tissue-type specificity and are localized within the cytoplasm and organelles such as nuclei, plastids and mitochondria.3Go The expression specificity and subcellular compartment of isoforms contribute to their diverse interactions with partners and differential functions in cellular activities. However, our understanding of the significance of 14-3-3 proteins in the regulation of plant development and environmental adaptation is still in its infancy.

Rice, with its relative small genome and important staple food crop, has been adopted as a monocotyledonous model plant for dissecting genetic networks of biotic and abiotic stress responses in cereal crops. The completion of the rice genome project allows genome-wide searching for the 14-3-3 family in the genome,27Go which has been found involved in rice stress responses as well as development. For example, the rice 14-3-3 proteins, GF14b, GF14c, GF14e and GF14f (previously named SR14-3-3), interact with target proteins that are involved in stress responses.28Go Interestingly, GF14b and GF14f may interact with mitogen-activated protein (MAP) kinase BIMK1, a component induced by rice blast fungus and known to participate in systemic acquired resistance.28Go In a large-scale microarry analysis for rice genes involved in defense responses, we identified some 14-3-3 genes were regulated by different interactions between rice and the pathogens or by defense signaling compounds. In this report, we analyzed the phylogenetics of the rice 14-3-3 family comprising eight members that are all expressed. We systemically analyzed expression patterns of four 14-3-3 genes that were differentially regulated by the rice blast fungus Magnaporthe grisea (M. grisea) and bacterial blight Xanthomonas oryzae pv. oryzae (Xoo), defense signaling compounds and diverse abiotic stress stimuli. We also observed subcellular locations of these isoforms.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussions
 Acknowledgements
 References
 
2.1. Mining of rice14-3-3s and phylogenetic analysis
Rice 14-3-3s were searched from the whole rice genome (japonica Nipponbare) with the conversed 14-3-3 protein sequence via the public GenBank database (http://www.ncbi.nlm.nih.gov/). The retrieved cDNAs or coding regions of 14-3-3s then were searched for their full-length cDNAs in the KOME database (http://cdna01.dna.affrc.go.jp/cDNA/). Genomic structures of the rice 14-3-3 genes were analyzed by comparing the cDNA sequences and the corresponding genomic DNA sequences. ClustalX 1.81 and Genedoc were used to multi-align nucleotide acid and amino acid sequences of the 14-3-3s. ClustalX 1.81 and TreeView 1.6.1 were used to perform phylogenetic analysis.

2.2. Plant growth and inoculation
A pair of rice (Oryza sativa L. ssp. indica) near isogenic lines, H7R (resistant to rice blast, with the resistance gene Pi-k/Pi-r1(t) from the donor Tetep) and H7S (susceptible) were grown in a growth chamber under 28°C/22°C and 14 h/10 h (day/night) for 2 weeks, then were spray-inoculated with M. grisea race ZB1 as described.29Go Rice suspension cells were made from the transgenic rice line 106 expressing the resistance gene Xa21 against Xoo30Go and were co-incubated with 107 cells per ml of the incompatible Xoo race P6 (99A) and the compatible race K1 (J18) as described.31Go Inoculated leaves/cells were harvested at a time course of 0–24 h postinoculation. All tissues were washed to remove surface microbes and frozen in liquid nitrogen immediately then stored at –80°C for RNA preparation.

2.3. Treatments with defense signaling compounds
Two-week-old seedlings of H7R were spray-treated with 300 µM benzothiadiazole (BTH, a salicylic acid analog), 100 µM methyl jasmonate (MeJA), and 100 µM ethephon (ETH, a precursor of ethylene), in the closed pots at a growth chamber. Water-spray was used as mock controls. Leaves were collected at 0, 4, 8, 12 and 24 h after treatments. The rice suspension cells were incubated for 0, 2, 6 and 12 h with various concentrations of H2O2.

2.4. Treatments with abiotic stress
Two-week-old seedlings of H7R were cultured with 150 mM NaCl, 20% PEG 6000 for salt and drought stress treatments, and with 100 µM abscisic acid (ABA) solution for 0–48 h. For wounding treatment, leaves were cut into 2–3 cm pieces and kept in 50 mM potassium phosphate buffer (pH 7.0) for 0–48 h.

2.5. RNA preparation and northern blot
Total RNAs were isolated from cells by using TRIzol tragent according to the manufacturer's protocol (Waston Biot). Each 30 µg of total RNAs was separated on a 1% formaldehyde-agarose gel for capillary blotting onto Hybond nylon membrane (Amersham Pharmacia), and hybridized with probes derived from a 320 bp SacII/SfiI fragment from GF14b, a 420 bp SfiI fragment from GF14c, a 400 bp BclI/SfiI fragment from GF14e and a 350 bp Cfr13I/SfiI fragment from GF14f. Similarities between each probe and the other GF14 genes are <55% to ensure probe specificity.

2.6. Tissue-specific expression of GF14 genes
Total RNA was prepared from seedling leaves, leaves, roots, internodes and panicles followed by the first cDNA strand synthesis for RT–PCR using SuperScriptTM RT–PCR kit (Invitrogen). Primers were designed for GF14b (5'-AGGACATTGCTTTGGCTGAG-3' and 5'-TTACTGCCCCTCGCTGGAG-3'), GF14c (5'-CGTTTGACGAAGCCATCTCC-3' and 5'-CTAGTAGAACAGGAGAAGAATC-3'), GF14e (5'-GATATTGCCCTGGCAGAGTTG-3' and 5'-GAGATATCGGAAGTCCACAGC-3') and GF14f (5'-GATATTGCCCTGGCAGAGTTG-3' and 5'-TTAGTGGCCCTCTCCTTCAG-3'), to amplify the fragments of 300 bp (GF14b), 280 bp (GF14c), 240 bp (GF14e) and 300 bp (GF14f), respectively. The rice ubi-1 was used as the control.32Go

2.7. Subcellular localization of GF14 protein
GF14-YFP fusions were made by in-frame fusion of the full-length GF14 cDNAs with YFP (accession number AA048591 [GenBank] ). The fusion genes were inserted into the vector pCAMBIAC1301 driven by the 35S promoter and were bombard-transformed into the wild-type cultivar TP309 calli by using a helium biolistic device (Bio-Rad PDS-1000/He). Calli were grown and selected on medium supplemented with hygromycin (20 mg/L) to generate GF14-YFP transgenic calli. Transient expression of the GF14-GFP fusions in onion epidermal cells was performed as previously described.33Go The transgenic rice and onion cells were observed with a confocal laser scanning microscope (Zeiss LSM510). 4'-6-diamidino-2-phenylindole (DAPI) fluorescence staining was performed to detect the nuclei. The cells were dipped in 30% sucrose solution for 30 min to perform plasmolysis.

2.8. Analysis of cis-acting elements in promoter regions of GF14 genes
According to the full-length cDNA sequences, the putative promoter regions were retrieved by searching the matched genomic sequences of the genes in GenBank (http://www.ncbi.nlm.nih.gov/BLAST/), with 2000 bp upstream of the 5' termini of the full-length cDNAs. The cis-acting elements, such as W, GCC and GCC-like, ABRE and DRE/CRT boxes in the promoter regions were analyzed.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussions
 Acknowledgements
 References
 
3.1. The rice GF14 family and comparison with the Arabidopsis family
By taking advantage of the completion of the rice genome project, we found that this model crop has a total of eight 14-3-3 genes located to six chromosomes that all have matched full-length cDNAs, suggesting that these genes are all expressed (Fig. 1A). Following the previous report,28Go the members of the rice family are named GF14a through GF14h (G-box factor 14-3-3 homologs), with SR14-3-3 named as GF14f. These GF14 proteins share highly conserved structures (Fig. 1A). According to the 14-3-3 classification,4Go GF14g and GF14h belong to the {varepsilon} group, while the rest belong to the non-{varepsilon} group. The phylogenetic topology of the rice family compared with the Arabidopsis family reveals five groups (Fig. 1B), of which groups A–D are similar to those for the Arabidopsis family and other plant 14-3-3s.4Go GF14g and GF14h fall into group D with five Arabidopsis 14-3-3s. However, the majority of the rice GF14s (six out of eight) constitute an additional group E. This result suggests that separate gene duplication of the non-{varepsilon} group could occur before the differentiation of the two species. The richness of diversity in these 14-3-3s indicates divergent roles of the members in biological processes in rice as well as in Arabidopsis.


Figure 1
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  		Figure 1. Rice 14-3-3 gene structure and phylogenetic tree of rice and Arabidopsis 14-3-3 families. (A) Genomic structure of eight rice 14-3-3 genes. Individual exons are delineated by segmentation. Numbers show nucleotide lengths of exons. The accession numbers for full-length cDNA and BAC clones are indicated. (B) A phylogenetic tree shows the topology of the rice (GF14) and Arabidopsis (GRF) 14-3-3 families within five major groups, based on cDNA sequences. A, B, C group are Arabidopsis non-{varepsilon} groups. Group D belongs to {varepsilon} group. Note that the majority of rice GF14s are classified into group E (non-{varepsilon} group). Branch numbers represent as percentage of bootstrap values in 1000 sampling replicates.

 
3.2. GF14 genes induced during the early rice-M. grisea and rice-Xoo interactions
In a large-scale microarray analysis of rice genes involved in defense responses, we found >1000 genes regulated by pathogen infection and other stress stimuli (Li et al., unpublished). The microarray data are available in our rice genomics database (http://www.nlpmg.labs.gov.cn/Signal/Ricearray/Supplemental1.xls). Among these regulated genes, we found four of the GF14 genes, GF14b, GF14c, GF14e and GF14f. The four GF14s share high similarity (overall 85.5–95.4% identities) on their amino acid sequences (Fig. 2A), but comparatively low similarity (overall 34.8–51.7% identities) on cDNA sequences. We analyzed the induction of these genes in the early incompatible and compatible rice-M. grisea interactions. Northern blot analysis showed that all four GF14 genes were induced by the fungal pathogen in a time course of 0–24 h postinoculation (Fig. 2B). Similarly, these genes except GF14f were also induced in the rice-Xoo interactions (Fig. 2C). Furthermore, it was shown that these genes appeared to be earlier or stronger induced in the incompatible interactions than in the compatible interactions between rice and the pathogens (Fig. 2B and C), consistent with the well-recognized expression pattern of defense-related genes during the host–pathogen interactions.34Go


Figure 2
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  		Figure 2. Alignments of four rice GF14 isoforms and their induction during rice defense responses. (A) Protein sequence alignment of the four GF14 isoforms with overall 85.5–95.4% identity. (B) Expression patterns of the GF14 genes in the incompatible and compatible rice-M. grisea interactions in a time course of 0–24 h postinoculation. C12, control for 12 h; C24, control for 24 h. (C) Expression patterns of the GF14 genes in the incompatible and compatible rice-Xoo interactions in a time course of 0–24 h postinoculation. Independent experiments were carried out with similar results. C24, control for 24 h.

 
3.3. GF14 genes regulated by abiotic stress
It has been known that plant 14-3-3 proteins are involved in abiotic stress responses.11Go We further analyzed the expression patterns of these GF14 genes in responses to abiotic stress including salinity, drought and wounding. Northern hybridization indicated that these GF14 genes were differentially regulated by the stress stimuli (Fig. 3). The transcription of GF14b was rapidly induced at 2–4 h by the salt and PEG6000 (drought-mimic) treatments, then declined to basal levels. Similarly, GF14c and GF14e were also induced at the early stage of 2–8 h by salt and drought stresses, consistent with the observation that the transcription of GF14b, GF14e was induced by a variety of stress stimuli.28Go GF14f appeared to be induced by salinity and drought through the whole time course of 2–48 h. Wounding also induced the GF14 genes except GF14b with peak levels at 24 h after treatment. Interestingly, the transcription of GF14e was decreased at 48 h in wounding treatment (Fig. 3). These differential expression patterns of the GF14 genes tested in face of abiotic stresses implicate that they may play regulatory roles in environmental responses.


Figure 3
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  		Figure 3. Differential regulation of GF14 gene expression by abiotic stress. The transcripts of GF14b, GF14c, GF14e and GF14f were detected after salt (150 mM NaCl), drought (20% PEG6000) and wounding treatments as described. C48, control for 48 h.

 
3.4. GF14 genes regulated by defense and stress compounds
It has been known that the phytohormones, salicylic acid, jasmonic acid and ethylene are signaling compounds involved in plant defense,34Go and hydrogen peroxide (H2O2), one of reactive oxygen intermediates resulting from an oxidative burst during the plant HR, acts as an activator of plant defense.35Go We systemically analyzed the induction of the four GF14 genes by these molecules. As shown in Fig. 4A, GF14f was induced by BTH, ETH and MeJA, similar was GF14e except it was first slightly repressed at 4 and 8 h by BTH. GF14b was slightly induced by BTH and ETH but not by MeJA. Similarly GF14c was significantly induced at 24 h by ETH and slightly induced by BTH and MeJA. Because of no consistent information for H2O2 treatment of rice cells, we tested the effect of different H2O2 concentrations on the induction of the 14-3-3 genes. All of the four GF14 genes were up-regulated by H2O2 even with low concentration (Fig. 4B). Low H2O2 concentration (0.01 mM) seemed to be more efficient to activate GF14c.


Figure 4
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  		Figure 4. Differential induction of GF14 genes by defense and stress molecules. (A) BTH (300 µM), MeJA (100 µM) and ETH (100 µM) differentially induced GF14b, GF14c, GF14e and GF14f. (B) H2O2 induced the GF14 genes. Note that low H2O2 concentration (0.01 mM) seemed to stronger induce GF14c. (C) Induction of the GF14 genes by ABA (100 µM). C48, control for 48 h.

 
ABA acts as a stress-responsive hormone and involves in modulating tolerance to drought and salinity.36Go Our analysis showed that ABA induced GF14e and GF14f during the time course (Fig. 4C). Similar to the observation in the salt and drought treatments (Fig. 3), GF14b and GF14c were obviously induced by ABA only within early 2–8 h of treatments (Fig. 4C). In addition, GF14c appeared to be more subject to environmental regulation, because even its basal expression level was somewhat different among independent treatments (Figs 2B and C,3 and 4).

3.5. Cis-elements in promoter regions of GF14 genes
It has been known that the cis-acting elements, W, GCC and GCC-like boxes, play important roles in expression regulation of defense-related genes,37Go and that the major cis-elements involved in abiotic stress response are ABRE and DRE/CRT.36Go,38Go We systematically analyzed these elements in the predicted 2.0 kb promoter regions of the four GF14 genes (Table 1). The analysis shows that the ABRE, W and GCC/GCC-like sequences are predominant elements in the predicted promoters of the GF14 genes analyzed, indicating their features of biotic and abiotic stress-mediated induction. We also found that the same cis-elements are present in the other four GF14 genes (Table 1). The biological significance of these elements in the regulation of gene expression is yet to be investigated.


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  		Table 1. Stress-responsive cis-elements in promoter regions of GF14 genesa

 
3.6. Tissue-specific expression and subcellular localization of GF14 proteins
We further analyzed the tissue-specific expression patterns of the GF14 genes, and found that these GF14s exhibited a degree of tissue specificity (Fig. 5). GF14e was expressed in all tissues tested, with the highest expression level in the panicle, similar was the expression pattern of GF14f except its low expression level in the flag leaf and internode of the heading plant. Whereas the transcript of GF14b was hardly detected in the flag leaf, internode and panicle of the heading plant, with higher level detected in the root. Similarly, the transcript of GF14c was hardly detected in the leaf and root of the heading plant, with the highest level detected in the seedling leaves. These tissue-specific patterns of GF14s' expression are probably associated with their differential functions.


Figure 5
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  		Figure 5. Tissue-specific expression and subcellular localizations of the GF14 isoforms. Expression of the GF14 genes in different tissues, detected by RT–PCR with the rice ubi-1 as a control. SL, seedling leaf; L, R, I, P for flag leaf, root, internode, panicle of heading plants, respectively.

 
14-3-3 proteins are widely localized in diverse subcellular compartments, implicating their multifunctional roles in cellular processes.3Go We made GF14-YFP fusion constructs and expressed them in rice cells. Because localization of YFP/GFP fusion proteins is usually obscure in rice callus, the cytoplasmic location of the fusion proteins was difficult to be imaged in the rice cells. We could observe obvious YFP fluorescence only in the compartments where the fusion proteins accumulated (Fig. 6A). With DAPI fluorescence probing to localize the nuclei, we observed that GF14b-YFP, GF14e-YFP and GF14f-YFP accumulated in the nuclei. For a clear vision of the localization of the YFP fusion proteins, transient expression was performed in onion epidermic cells. This additional experiment clearly showed that GF14-YFP fusion proteins were all targeted to the cytoplasm (Fig. 6B). Same to the observation with the transformed rice calli, GF14b-YFP, GF14e-YFP and GF14f also accumulated in the nuclei. Similar subcellular localization was also observed for the Arabidopsis 14-3-3 proteins.3Go,4Go Hence, diversity in the expression and subcellular localization of the rice 14-3-3 proteins is present to ensure their functions in certain cells or tissues.


Figure 6
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  		Figure 6. Subcellular localizations of the GF14 isoforms in rice callus and onion epidermal cells. (A) Subcellular localizations of the GF14 isoforms in rice callus cells. (B) Subcellular localizations of the GF14 isoforms in onion epidermal cells. Plasmolysis was performed to confirm subcellular localization of the GF14s. YFP fluorescence (top right) was detected together with bright field images of cells (top left); DAPI fluorescence images (bottom left) and all images merged (bottom right). N, nucleus.

 

   4. Discussions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussions
Acknowledgements
 References
 
14-3-3 proteins, acting as ubiquitous regulators, have been known to play important roles in many plant biological processes. However, their involvement in abiotic and abiotic stress responses, two biological activities with agricultural significance, has not been well studied. In this paper, we have shown that the rice 14-3-3 genes, GF14b, GF14c, GF14e and GF14f, were induced differentially by the rice fungal and bacterial pathogens, defense signaling compounds and abiotic stress stimuli (Figs 2–4). Our current study adds to the previous findings of the rice 14-3-3 family28Go,39Go,40Go and would serve as a start point to explore their functions in disease resistance and stress tolerance in the model cereal crop.

It is intriguing that the rice 14-3-3 family has most likely only eight isoforms classified into 2 groups in comparison with 12 isoforms expressed in the Arabidopsis family (Fig. 1), given the much larger genome of rice than that of Arabidopsis. It has been known that, in spite of the conservation of their core structure, 14-3-3 isoforms have a degree of specificity with regard to the target proteins with which they interact.11Go It is also interesting that the rice 14-3-3 proteins except two constitute a separate group (group E) (Fig. 1B). A systemic and comparative analysis of the target proteins of the rice and Arabidopsis 14-3-3 isoforms would contribute to fundamental understanding of the conservation and divergence of biological processes between the two model plants.

We found that all of four GF14 genes studied were differentially regulated by the rice pathogen challenge and abiotic stress, indicating they may play important roles in rice defense and abiotic stress tolerance. Similar involvement of 14-3-3s in defense and stress responses has also been frequently observed in other plants. In addition to the evidence described above, a 14-3-3 gene is induced during the HR of soybean inoculated with P. syringae,41Go and in cotton inoculated with the wilt pathogen V. dahliae.42Go In tomato, ten 14-3-3 genes were found to be differentially regulated in resistant or susceptible plants challenged with the avirulence elicitor Avr9.18Go In ABA-mediated stress responses, 14-3-3 proteins are constituents of transcription factor complexes for ABA-induced gene expression.2Go Direct evidence for 14-3-3s involved in disease resistance or stress tolerance also comes from genetic studies. A quantitative resistance marker (QTL) against fungal pathogens is mapped onto a region of chromosome 4AL of wheat containing a 14-3-3 gene.43Go GF14c is localized within the interval of drought tolerance QTL oa8.1, and SR14-3-3/GF14f interacts with an ATPase known to locate to the region of drought tolerance QTL oa3.1.28Go,39Go More recently, GF14e (previously named as 14-3-3 protein-A) is localized in the QTL region for blast resistance on chromosome 2.40Go It has been extensively documented that transmembrane ion flux is involved in early defense signaling, therefore, a role for some 14-3-3 proteins in plant defense can be speculated, which probably interact with some membrane-bound clients such as H+-ATPase. Interestingly, GF14b and GF14c exhibited similar induction patterns during the salinity, drought and ABA treatments (Figs 3 and 4C), whether they play an important role in face of these stress conditions remains to be evaluated. We also found the cis-elements responsive to pathogen attack and abiotic stress in the rice GF14 genes (Table 1), appropriately explaining the features of the induction of the genes by biotic and abiotic stresses. Therefore, 14-3-3s are also subject to the regulation by certain transcript factors.

Given the size of 14-3-3 gene families and the wide range of pathways in which they have been involved, it would be difficult or trivial to identify the phenotypes of individual 14-3-3 mutations.4Go,8Go 14-3-3s act as scaffolds for the assembly of large signaling complexes, and various 14-3-3 isoforms have different affinities for at least certain target proteins that are most likely with the same cellular localization for functioning. Complete understanding of 14-3-3 roles will require the determination of cellular and subcellular localizations and targets of specific isoforms. For example, GF14c is associated with at least 10 proteins known to accumulate in the thylakoid.28Go Therefore, searching for and identifying functions of the target proteins of a given 14-3-3 through the forward or reverse genetic approach would provide a clue for the 14-3-3-mediated cellular activities.


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussions
 Acknowledgements
References
 
This work was supported by the National Natural Science Foundation of China (30125030), the Ministry of Science and Technology of China (2002AA2Z1003) and the Chinese Academy of Sciences project (KSCX2-SW-301-02).


   Footnotes
 
*To whom correspondence should be addressed. Tel. +86-21-54924121, Fax. +86-21-54924015, E-mail: zhhe{at}sibs.ac.cn

Communicated by Satoshi Tabata


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussions
 Acknowledgements
 References
 

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