Supported by the State Key Basic Research and Development Plan of China (2005CB120901) and Science and Technology Department of Zhejiang Province, China.
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The rice (Oryza sativa L.) genome contains at least six genes exclusively with an SPX (SYG1/PHO81/XPR1) domain at the N-terminal, designated as OsSPX1-6. Here we report the diverse expression patterns of the OsSPX genes in different tissues and their responses to Pi-starvation. Among them, five genes, OsSPX1, 2, 3, 5 and 6 are responsive to Pi-starvation in shoots and/or in roots. The subcellular localization analysis indicates that OsSPX1 and OsSPX2 is exclusively located in nucleus, OsSPX3 in the cytoplasm, and OsSPX4 is a membrane localization protein. OsSPX1 regulates OsSPX2, 3 and 5 at the transcription level and is positively involved in the responses of the genes to Pi-starvation. Overexpression of OsSPX3 downregulates OsSPX5 in shoots under Pi-sufficiency. OsSPX3 negatively regulates the PSI (Pi-starvation induced) gene, OsIPS1 and is involved in the responses of miR399 and OsPHO2 to Pi-starvation. Our results suggest that OsSPX1 may be a regulator involved in the transcriptions of OsSPX2, 3 and 5. OsSPX3 plays a role in OsIPS1/miR399 mediated long distance regulation on OsPHO2. Our results also indicate that OsSPX3 is involved in plant tolerance to Pi-starvation stress.
The SPX (SYG1/PHO81/XPR1) domain at the N-termini of various proteins was defined following that of SYG1 (suppressor of yeast gpa1), PHO81 (cyclin-dependent kinase) in yeast (Spain et al. 1995; Lenburg and O'Shea 1996) and the human protein XPR1 (the xenotropic and polytropic retrovirus receptor) (Battini et al. 1999). ScPHO81 is a cyclin-dependent kinase (CDK) inhibitor that is induced by Pi starvation, interacting with cyclin ScPHO80 to repress the activity of CDK ScPHO85, thus promoting the expression of ScPHO5 and enhancing yeast tolerance to Pi starvation (Lenburg and O'Shea 1996). More recently, the function of ScPHO81 as a critical protein for external phosphate sensing and for signaling through the low orthophosphate affinity carriers has been known (Pinson et al. 2004). Several SPX domain genes in plants were found to be involved in responses to environmental cues or internal regulation of nutrition homeostasis. Barley IDS4 (iron-deficiency specific clone 4) contains part of the SPX domain and is preferentially expressed in Fe-deficient roots (Nakanishi et al. 1993). Arabidopsis PHO1, harboring both SPX and EXS (ERD1/XPR1/SYG1) domains, plays a role in loading root Pi into the xylem vessels. Loss of AtPHO1 function in pho1 mutants results in Pi deficiency in above-ground tissues (Poirier et al. 1991; Hamburger et al. 2002; Wang et al. 2004). AtSHB1 (SHORT HYPOCOTYL UNDER BLUE 1) protein containing SPX and EXS domains has been reported to be a sensor of blue light and involved in seed development (Kang et al. 2006; Zhou et al. 2009).
Twenty genes in Arabidopsis with the SPX domain were identified based on Arabidopsis genome sequence data (Wang et al. 2004). The 20 genes were grouped into four sub-families. Three sub-families, with a total of 16 members, encode proteins with the SPX domain and an extra conservative domain. The other four members (At5g20150, At2g26660, At2g45130 and At5g15330) form a unique sub-family with no conservative region of the Pfam-A type other than the SPX domain. Recently, four genes not encoding any conservative region other than a SPX domain in Arabidopsis were characterized and were found to have diverse functions in plant tolerance to phosphorus starvation under the AtPHR1 regulation system (Duan et al. 2008). Repression of AtSPX3 by RNA interference led to aggravated phosphate (Pi)-deficiency symptoms, altered P allocation and enhanced expression of a subset of phosphate-responsive genes including AtSPX1. These findings suggest that the SPX domain proteins may play important roles on Pi-signaling in a network system.
Recently, the molecular components involved in Pi signaling pathways has been reviewed (Yuan and Liu 2008; Valdés-López and Hernández 2008). Four transcription factors, PHR1, WRKY75, ZAT6 and BHLH32, involved in P starvation signaling have been characterized in Arabidopsis. In rice, two homologs of the Arabidopsis PHR1 gene, OsPHR1 and OsPHR2, were identified to be involved in Pi starvation signaling pathway by regulation of the expression of PSI genes (Zhou et al. 2008). OsPHR1 and OsPHR2 positively regulate OsIPS1 for a non-coding RNA. IPS1 sequesters miR399 (microRNA 399), which negatively regulates the gene PHO2 at the post-transcriptional level (Fujii et al. 2005; Aung et al. 2006; Bari et al. 2006; Chiou et al. 2006; Franco-Zorrilla et al. 2007). PHO2 is downregulated by Pi-starvation, which is involved in the enhancement of uptake and translocation of plants. Although the Pi-signaling mediated by PHR-IPS1-miR399-PHO2 system is well established, the molecular network on the system is still to be elucidated.
At least six genes with an exclusive SPX domain in rice were identified based on the present rice genome database (http://www.ncbi.nlm.nih.gov/tBLASTn/). Among the six genes, OsSPX1 negatively regulates Pi accumulation in shoots and is involved in Pi-starvation signaling (Wang et al. 2008). To evaluate the potential functions of the other OsSPXs and their effects on Pi-signaling and the regulation of Pi uptake and accumulation, the transgenic plants with overexpression of OsSPX3 were developed. Our results indicate that OsSPX1, as a nucleus-localization protein, may be a regulator involved in transcriptions of OsSPX2, 3 and 5, and OsSPX3 may play a role in OsIPS1/miR399 mediated long distance regulation on OsPHO2.
Analysis of OsSPX gene and protein structures
For the six OsSPX genes with exclusive SPX domain, the full length cDNAs of four genes (designated as OsSPX1 (LOC_Os06g40120), OsSPX2 (LOC_Os02g10780), OsSPX3 (LOC_Os10g25310), and OsSPX4 (LOC_Os03g61200) are available (http://www.tigr.org). The gene structures were predicted for OsSPX5 (LOC_Os03g29250) and OsSPX6 (LOC_Os07g42330) using the GeneMarkHMM predictions program (http://www.tigr.org/tigr-scripts/osa1_web/gbrowse/rice/) based on the expressed sequence tags (ESTs) of the genes. The gene structure analysis showed three exons and two introns in OsSPX1-4 and OsSPX6, while in OsSPX5 there are only two exons and one intron. The exon distribution is similar in OsSPX1-3 with the larger second intron in OsSPX1 than in OsSPX2 and OsSPX3. The first intron in OsSPX4 and OsSPX6 is larger than those in other genes (Figure 1A).
The deduced proteins of the six genes showed similar lengths (247–320 amino acids) with 31–69% similarity (Figures 1B, 2). Analysis of their tripartite SPX domain revealed 40–72% similarity of amino acids in the entire SPX domain, but >80% in each subdomain. Amino acid sequence alignment analysis indicates that the SPX domain in rice and in Arabidopsis is highly conserved (Figure 2). Phylogenetic analysis for the SPX domain proteins of rice (Oryza sativa L.) and Arabidopsis, IDS4 protein in barley (Hordeum vulgare) and tomato (Lycopersicon esculentum), ADL143Wp in cotton (Ashbya gossypii) and PHO81 in yeast (Saccharomyces cerevisiae) indicates that OsSPX members can be grouped into three subgroups. OsSPX4 is in the first subgroup with AtSPX4, ScPHO81, AgADL and HvIDS-4, the second subgroup including OsSPX3, OsSPX5 and OsSPX6 closed to AtSPX3, and the third subgroup contains OsSPX1 and OsSPX2 with LeIDS4-like, AtSPX1 and AtSPX2. The results indicate the conservation of the SPX domain genes between monocot and dicot species (Figure 1C).
Tissue expression patterns and responses of OsSPX genes to Pi-starvation
Semi-quantitative reverse transcription–polymerase chain reaction (RT-PCR) was used to monitor transcript levels of the OsSPX genes in different tissues. To ensure specificity, the primer sequences were aligned with the rice genome (Table 1). Except for OsSPX5, the expression was detected only in roots and callus, a constitutive expression pattern was observed in five other genes (Fig. 3A). The transcript level of OsSPX1-3 was higher in root, leaf and callus than in stem, panicle and seed. The transcript level of OsSPX6 in root and leaf was much higher than in other tissues. RT-PCR analysis indicates that except for OsSPX4, other SPX genes were induced by Pi-starvation in shoot and/or in root (Figure 3B). To investigate the tissue expression patterns of the genes, the 2.0 kb promoter/5′ untranslated region (UTR) of OsSPX1, OsSPX2 and OsSPX5 were amplified and fused to the report gene for β-glucuronidase (GUS) in pCambia1391Z and then transformed into rice (Nipponbare). The GUS staining indicated that, the three genes were expressed in lateral roots and lateral root primordia, and the expression was enhanced by Pi-starvation (Figure 4A–F).
Table 1. The primers used for reverse transcription-polymerase chain reaction (RT-PCR) analysis, cloning of promoters and OSPX1-4 genes for subcellular localization and development of transgenic plants
For RT-PCR analysis:
For cloning of promoters:
For cloning of genes:
Subcellular localization of the OsSPX proteins
To determine the SPX protein localizations, C-terminal green fluorescent protein (GFP) fusions driven by CaMV 35S were used for visualization of OsSPX1-4 localizations. Expression clones were transformed into onion epidermal cells for transient assay. OsSPX1 and OsSPX2 were localized exclusively to the nucleus, and OsSPX4 to membrane (Figure 4H,I,K), while fluorescence of OsSPX3-GFP was present in onion cells as many fluorescent speckles in the cytoplasm (Figure 4J). The different localizations of the OsSPX proteins imply the different roles of the genes.
OsSPX1 regulates OsSPX2, OsSPX3 and OsSPX5 at the transcription level
OsSPX1 is involved in Pi-starvation signaling indicated by PSI genes (Pi-starvation induced) (Wang et al. 2008) and in addition to OsSPX1, four SPX genes were found to be responsive to Pi-signaling (Figure 3B). To determine whether OsSPX1 is involved in the responses of other OsSPX genes to Pi-signaling, the expression patterns of the OsSPX genes in roots and shoots of the transgenic plants of OsSPX1 were investigated using RT-PCR and qRT-PCR analysis. The PSI gene, OsIPS1, was used as an indicator for Pi-starvation signaling (Hou et al. 2005). Under Pi-sufficient conditions, both overexpression and repression of OsSPX1 downregulated OsSPX2 in shoots. In roots OsSPX3 and OsSPX5 were downregulated by overexpression of OsSPX1, and OsSPX2 was downregulated by repression of OsSPX1 (Figure 5A,B). Under Pi-starvation, overexpression of OsSPX1 aggravated the responses of OsSPX3 and OsSPX5 to Pi-starvation, and repression of OsSPX1 reduced the responses of OsSPX2, 3, 5 and OsIPS1 to Pi-starvation in shoots. In roots, repression of OsSPX1 downregulated OsSPX2 and OsSPX3, and reduced the responses of OsSPX5 and OsIPS1 to Pi-starvation (Figure 5A,B). The results indicate that OsSPX1 regulates the expression of OsSPX2, 3 and 5 which are tissue- and Pi-dependent.
OsSPX3 downregulates OsIPS1 and suppresses the responses of OsmiR399 and OsPHO2 to Pi-starvation
The RT-PCR and qRT-PCR analysis showed that OsSPX3 downregulates OsIPS1 (Figure 5C,D). It has been known that the non-coding RNA IPS1 sequesters miR399 (microRNA 399), which regulates the gene PHO2 at the post-transcriptional level (Fujii et al. 2005; Aung et al. 2006; Bari et al. 2006; Chiou et al. 2006; Franco-Zorrilla et al. 2007). We then investigated the expression pattern of OsPHO2 and precursors of OsmiR399d and OsmiR399j in the transgenic plants with overexpression of OsSPX3. The results showed that the repression of OsPHO2 and induction of OsmiR399 in roots by Pi-starvation was suppressed in OsSPX3 overexpressed plants (Figure 5D), indicating that OsSPX3 may be a repressor involved in the response of OsPHO2 to Pi-starvation mediated by OsIPS1/OsmiR399 regulation. The inductions of OsmiR399d and OsmiR399j by Pi-starvation were also suppressed in shoots of OsSPX3 overexpressed plants. Overexpression of OsSPX3 also downregulates OsSPX5 in shoots under Pi-supplied conditions (Figure 5D).
Overexpression of OsSPX3 inhibits plant growth
To evaluate the potential function of OsSPX3, the growth performance and Pi concentration in leaves and roots of the transgenic plants with overexpression of OsSPX3 were investigated in a solution culture with Pi-sufficient (10 mg Pi/L) and Pi-deficient (0.5 mg Pi/L) conditions. The experiment showed that overexpression of OsSPX3 inhibited plant growth under both Pi-sufficient and Pi-deficient conditions, but more severely under Pi-deficient conditions (Figure 6). Under Pi-sufficient conditions, the dried biomass of shoots and roots of the transgenic plants with overexpression of OsSPX3 was decreased by about 55% compared with that of the wild type, while it was decreased by about 70% under Pi-deficient conditions (Figure 6C,D). No significant difference in Pi concentration was observed between the transgenic plants and the wild type plants under both Pi-sufficient and Pi-deficient conditions (data not shown). The results suggest that OsSPX3 is involved in tolerance to Pi-starvation, but not in Pi accumulation.
The SPX domain genes are highly conservative in plants. In rice we found at least six genes exclusively with the SPX domain and five genes among them are responsive to Pi-starvation, indicating that the SPX domain genes are involved in the Pi-signaling network in plants. Although the OsSPX genes are similar in structure and sequence, various expression patterns of the genes and their different responses to Pi-starvation signaling imply the functional diversification of the genes. The different subcellular localizations of these proteins support the inferable conclusion.
Four Arabidopsis genes (designated as AtSPX1-4) exclusively with SPX domain have been characterized and their diverse functions in plant tolerance to Pi-starvation were revealed. Partial repression of AtSPX3 resulted in a decrease in tolerance to Pi-starvation (Duan et al. 2008). Similar gene structure of the SPX genes were found between rice and Arabidopsis with three exons and two introns, except for OsSPX5, which has only two exons and one intron. Compared with the gene structures of SPX1-4, the protein structures are more divergent between AtSPXs and OsSPXs. The Pfam B domain is found in AtSPX1, AtSPX2 and AtSPX4, but only in OsSPX4. AtSPX1 and AtSPX2 are not clustered closely to OsSPXs indicated by phylogenetic analysis (Figure 1). OsSPXs can be classified into three clusters: one OsSPX1-OsSPX2 pair cluster, one OsSPX3, OsSPX5, OsSPX6 and AtSPX3 cluster, and one cluster including OsSPX4-AtSPX4 pair, HvIDS4, AgADL and ScPHO81. OsSPX4 is membrane localized protein (Figure 4). Whether OsSPX4 functions as ScPHO81 in Pi-signaling pathway on plant Pi-starvation tolerance is an interesting question. It is notable that although AtSPX4 is highly homologous with OsSPX4, AtSPX4 is negatively responsive to Pi-starvation (Duan et al. 2008). The difference in their roles in the Pi-signaling system remains to be elucidated. It has been demonstrated that ancestral genome duplication occurred in cereals before evolutionary divergence of cereals (Goff et al. 2002; Guyot and Leller 2004). The phylogenetic analysis supports the hypothesis that SPX4 is more ancestral in the evolution history, and OsSPX5 and OsSPX6 may be duplicated from ancestral SPX3 with the change of gene structure.
Among the four AtSPX genes, AtSPX1-3 was upregulated by Pi-starvation in both shoots and roots, while AtSPX4 was downregulated (Duan et al. 2008). The Pi-starvation regulated expression patterns are similar for SPX1 and SPX3 between Arabidopsis and rice. The similar responsive pattern implies the conservative function of SPX1 and SPX3 genes in Arabidopsis and rice. Partial repression of AtSPX3 by RNA interference led to aggravated Pi-deficiency symptoms, altered Pi allocation and enhanced expression of a subset of phosphate-responsive genes including AtSPX1 (Duan et al. 2008). Our data showed that overexpression of OsSPX3 led to inhibition of plant growth under both Pi-supplied and Pi-deficient conditions, but more severely under Pi-deficient conditions. The results suggest that the function or the regulation system of SPX3 may be different between rice and Arabidopsis.
It has been demonstrated that OsSPX1 is involved in the Pi-signaling pathway and is a negative regulator for Pi accumulation in shoot tissues at high Pi levels (Wang et al. 2008), which is under regulation of OsPHR2, a central regulator of Pi-signaling (Rubio et al. 2001; Schachtman and Shin 2007; Zhou et al. 2008). The present data indicates that OsSPX1 is positively involved in the responses of OsSPX2, 3 and 5 to Pi-starvation signaling, and differently regulates OsSPX2, 3 and 5 in shoot and root. OsSPX2 is regulated by homeostasis of OsSPX1 in shoot and is remarkably downregulated by repression of OsSPX1 in root under Pi-supplied and Pi-starvation conditions (Figure 5). In root, OsSPX1 also negatively regulates OsSPX3, 5 and OsIPS1 under Pi-supplied conditions. OsSPX1 is a nucleus localization protein (Figure 4). Whether and how OsSPX1 regulates OsSPX2, 3 and 5 requires further investigation. In addition, repression of OsSPX1 results in Pi excessive accumulation in shoot (Wang et al. 2008) and OsSPX2 is downregulated by repression of OsSPX1 in root. Therefore, it is also an interesting question whether OsSPX2 can be involved in the regulation of OsSPX1.
In Arabidopsis, miR399 activity is inhibited by the non-protein coding gene AtIPS1 through a target mimicry mechanism (Franco-Zorrilla et al. 2007) which is downstream of AtPHR1. AtmiR399 reciprocally regulates the gene AtPHO2 at the post-transcriptional level (Fujii et al. 2005; Chiou et al. 2006). AtPHO2 functions as an ubiquitin-conjugating E2 enzyme (UBC24), and loss of function of PHO2/UBC24 will lead to excessive accumulation of Pi in the shoot tissue (Aung et al. 2006; Bari et al. 2006). The regulatory mechanism of miR399 and PHO2 is conserved in Arabidopsis and rice (Bari et al. 2006). Overexpression of OsSPX3 negatively regulates OsIPS1 and suppresses the reduction of OsPHO2 in roots by Pi-starvation (Figure 5). A regulatory network of AtmiR399 and AtPHO2 by long-distance signaling has been proposed (Lin et al. 2008; Pant et al. 2008), whereby the long-distance movement of miR399s from shoots to roots is crucial to enhance Pi uptake and translocation during the onset of Pi deficiency. Our data show that although OsIPS1 was downregulated in both shoots and roots, and the induction of OsmiR399 was suppressed in both shoots and roots, and repression of OsPHO2 under Pi-starvation was only detected in roots. These results suggest that OsSPX3 is involved in the systemic regulatory network of OsIPS1, OsmiR399 and OsPHO2.
Overexpression of OsSPX3 inhibits plant growth under both Pi-sufficient and deficient conditions, but more severely under Pi-deficient conditions (Figure 6). Under Pi-deficiency, repression of OsPHO2 in roots enhances Pi uptake and translocation. In OsSPX3 overexpressed plants, OsPHO2 cannot be repressed, which could at least partially explain the more severe inhibition of the transgenic plants with overexpression of OsSPX3 under Pi-deficiency. Of note, the growth of OsSPX3 overexpressed plants was also inhibited under Pi-supplied conditions, and therefore, another function of OsSPX3 on plant growth could be reasoned. In fact, overexpression of OsSPX3 downregulates OsSPX5 in shoots. Elucidation of the function of OsSPX5 may help us to understand the regulatory mechanism of OsSPX3.
In summary, six OsSPX genes with exclusive SPX domain were identified in rice. Five of the genes are responsive to Pi-starvation in shoot and/or in root, indicating that the subset of SPX genes is involved in the Pi-signaling network in a complex regulatory system (Figure 7). OsSPX1, as a nucleus localization protein, regulates transcriptions of OsSPX2, 3 and 5. OsSPX3 negatively regulates OsIPS1 and may be involved in the systemic regulatory network of OsIPS1, OsmiR399 and OsPHO2. OsSPX3 also functions on plant growth.
Materials and Methods
Phylogenetic and gene structure analysis
SPXs sequences from Arabidopsis (Arabidopsis thaliana L.) and rice (Oryza sativa L.) were identified through a BLAST search of The Arabidopsis Information Resource (TAIR; http://www.arabidopsis.org) and The Institute for Genomic Research (TIGR) (http://www.tigr.org) databases using AtSPX genes as a query (Duan et al. 2008). Data obtained were predicted amino acid sequences based on EST clones. Sequences were edited and aligned using EditSeq and Megalign (Lasergene DNASTAR) software. Multiple alignments were prepared using ClustalX 1.81 (Thompson et al. 1997). Neighbor-joining phylogenetic trees were generated using Tree-View 1.6.6 (http://darwin.zoology.gla.ac.uk/wrpage/treeviewx).
Plant materials and growth conditions
The japonica variety Nipponbare was used for all experiments and rice transformation. Hydroponic experiments were conducted using normal rice culture solution containing 1.425 mM NH4NO3, 0.323 mM NaH2PO4, 0.513 mM K2SO4, 0.998 mM CaCl2, 1.643 mM MgSO4, 0.009 mM MnCl2, 0.075 mM (NH4)6Mo7O24, 0.019 mM H3BO3, 0.155 mM CuSO4, 0.036 mM FeCl3, 0.070 mM citric acid, and 0.152 mM ZnSO4 (Yoshida et al. 1976). Rice plants were grown in a growth room with a 12:12 h light : dark (LD) cycle (200 μmol/m2 per s photon flux density) and a day/night temperature of 30/22 °C. The high and low Pi level was treated using 10 mg Pi/L and 0.5 mg Pi/L, respectively. Pi-starvation condition was created under solution culture with no Pi for 7 d for 7-d-old seedlings. For RT-PCR and qRT-PCR analysis of gene expression, 14-d-old seedlings were used for sampling of roots and shoot tissues.
Semiquantitative RT-PCR and quantitative RT-PCR
First-strand cDNAs were synthesized from total RNA using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Semiquantitative RT-PCR was carried out using a pair of gene-specific primers. The PCR products were loaded on 1.2% agarose gels and photographed using a charge coupled device (CCD) camera. The levels of gene expression were compared using the densities of the PCR products as a basis. The housekeeping gene ACTIN was used as an internal control. Real-time qRT-PCR was carried out using an SYBR PremixEx Taq (Perfect Real Time) Kit (TaKaRa Biomedicals, Tokyo, Japan) on a LightCycler480 machine (Roche Diagnostics, Basel, Switzerland), according to the manufacturer's instructions. The amplification program for SYBR Green I was carried out at 94 °C for 10 s, 58 °C for 15 s, and 72 °C for 22 s. Triplicate quantitative assays were carried out on each cDNA sample. The relative level of expression was calculated using the formula 2−Δ(ΔCp). All of the primers that were used for the RT-PCR are given in Table 1.
Promoter–GUS constructs and histochemical analysis
The promoter 5′ UTR regions of the OsSPX1, OsSPX2 and OsSPX5 were amplified from genomic DNA of japonica variety Nipponbare using the primer pairs shown in Table 1. PCR products were first cloned into the pUCm-T vector (Shenerg Biocolor Co. Ltd; http://www.biocolors.com.cn) and sequenced for confirmation. Promoter fragments with correct insertion orientation were then transferred from pUCm-T to the expression vector pCambia1391Z. The resultant promoter–GUS constructs and the original pCambia1391Z were transformed into japonica variety Nipponbare. Histochemical GUS staining was carried out as described previously (Jefferson et al. 1987). To study the promoter activity in response to Pi starvation, transgenic seeds were grown in solution culture for 10 d and exposed to Pi treatment for another 10 days.
Subcellular localization analysis
Full-length cDNA clones for the OsSPX1-4 genes were directly amplified from rice root cDNAs (http://www.tigr.org) using the primer sets shown in Table 1 and digested with SpeI and BglII/NcoI. The gene fragments were introduced into pCambia1302 linearized with SpeI and BglII/NcoI. The resultant constructs were confirmed by PCR and restriction analysis. All of the GFP fusion constructs, together with original pCambia1302, were transiently expressed in onion epidermal cells using the Bio-Rad biolistic PDS-1000/He system (http://www.bio-rad.com/), carried out essentially as described previously (Varagona et al. 1992). The GFP fluorescence was imaged using a Carl Zeiss laser scanning system LSM510 (http://www.zeiss.com/).