Gene expression of jojoba (Simmondsia chinensis) leaves exposed to drying

Available online at www.sciencedirect.com

Environmental and Experimental Botany 63 (2008) 137–146

Gene expression of jojoba (Simmondsia chinensis) leaves exposed to drying Hongwei Geng a , Lu Shi a , Wei Li a , Bo Zhang a , Chengcai Chu a,b , Hongjie Li c , Genfa Zhang a,∗ a College of Life Sciences, Beijing Normal University, Beijing 100875, China Institute of Genetics and Development Biology, Chinese Academy of Sciences, Beijing 100101, China c College of Life and Environmental Sciences, Central University for Nationalities, Beijing 100081, China b

Received 5 February 2007; received in revised form 7 September 2007; accepted 6 October 2007

Abstract Jojoba (Simmondsia chinensis (Link) Schnieder) was used to identify genes regulated by wound–water stress. Suppression subtractive hybridization (SSH) was performed using cDNAs prepared from wounded parts of leaves under drying stress as a tester and cDNAs from unstressed parts of leaves as a driver. A forward-subtracted cDNA library was constructed and positive clones were confirmed by differential screening, resulting in 1344 clones as wound–water stress induced. After sequencing and trimming, 838 sequences were further analyzed. Sequence assembly analysis generated 385 unique ESTs. By referring to NCBI database and the functional categories of Arabidopsis thaliana proteins, 139 ESTs in 13 main categories were annotated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to evaluate the functions of the ESTs and the pathways in which they are involved. Ninety-six genes were identified by KEGG Orthology (KO) identifier. These genes are involved in 63 pathways. Some pathways, such as energy metabolism, lipid metabolism, amino acid metabolism, translation, and MAPK signaling pathway, are associated with wound–water stress. The results from this study are useful in understanding the genetic regulation process under wound–water stress in jojoba. © 2008 Elsevier B.V. All rights reserved. Keywords: Jojoba; Wound–water stress; Suppression subtractive hybridization

1. Introduction Water deficit is one of the most common environmental stresses that limit growth and productivity of agronomically important crops. Plants suffer from water-stress not only under drought and high salinity conditions but also under low or high temperature. Water-stress induces various biochemical and physiological responses in plants that ensure them to survive (Bohnert et al., 1995; Bray, 2002). Tolerance of plants to water deficit is determined by multiple biochemical pathways that facilitate retention and/or acquisition of water, protect chloro-

Abbreviations: ESTs, expressed sequence tags; SSH, suppression subtractive hybridization; MIP, major intrinsic protein; PIP, plasma membrane intrinsic protein. ∗ Corresponding author at: College of Life Sciences, Beijing Normal University, #19 XinWai Street, Beijing 100875, China. Tel.: +86 10 5880 9453; fax: +86 10 5880 7721. E-mail address: [email protected] (G. Zhang). 0098-8472/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2007.10.026

plast functions, and maintain ion homeostasis (Hoekstra et al., 2001; Ingram and Bartels, 1996; Shinozaki and YamaguchiShinozaki, 1997). Understanding of the details that plants perceive stress signals and transmit the signals to cellular machinery to activate adaptive responses is important in discovery of the mechanism of tolerance to stress. Cellular and molecular responses of plants to abiotic stresses have been studied mainly using Arabidopsis thaliana (Zhu et al., 1997). Zhu (2002) and Xiong et al. (2002) reviewed the signal transduction network of drought and other environmental stresses. Known signaling components and lots of signaling components with unknown functions comprise of a complex signal transduction network. Xiong et al. (2002) categorized signaling components and pathways into three signaling types: (1) osmotic/oxidative stress signaling that makes use of mitogen-activated protein kinase (MAPK) modules; (2) Ca2+ -dependent signaling that leads to the activation of LEA-type genes (e.g., the DRE/CRT class of genes); (3) Ca2+ -dependent SOS signaling that regulates ion homeostasis. Although the functions of some components

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and a few pathways have been explained, water-stress signaling transduction remains to be further studied. Although mutants from genetic model plants were commonly used to study water stress, seeds or resurrection plants and agriculturally relevant plants were also used as the target plant species (Ingram and Bartels, 1996). Jojoba (Simmondsia chinensis (Link) Schnieder) is an evergreen shrub. The commercial value of this species is the wax, which is a special oil only found in jojoba seeds (National Research Council, 1985). Jojoba is an extremely drought-tolerant plant species in the world (Benzioni and Dunstone, 1986). It is native to the deserts of the American Southwest, the most inhospitable land on the earth, where the annual precipitation in some regions of this area is as low as 80 mm, and temperature is as high as 54 ◦ C (Benzioni, 1995). In the past two decades, most studies on jojoba mainly focused on the following fields: (1) development of cultivars via selecting appropriate clones with higher seed yield and climate adaptation for commercial cultivation; (2) discovery of reliable and simple tissue culture methods for mass propagation in vitro; (3) physical and chemical characteristics of jojoba oil and its industrial applications; (4) characterization and use of byproducts of seeds after extracting jojoba oil. A few studies on cloning and expressing wax synthesis genes were reported (Michale et al., 1999, 2005). Suppression subtraction hybridization (SSH) is a powerful technique widely used in comparing two populations of mRNA to isolate clones of differentially expressed genes (Diatchenko et al., 1996; Ji et al., 2003). To understand the molecular basis of jojoba in response to water stress, we cloned wound–water stress-regulated ESTs in jojoba mature leaves by SSH and differential screening, and then analyzed their possible functions in response to water stress and the signal transduction pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Kanehisa et al., 2006). 2. Materials and methods 2.1. Materials and treatments Eight-year-old female jojoba plants were grown in greenhouse with enough water supplies. Mature leaves used as the starting materials were immediately cut into two parts. The upper part (no leafstalk) of each leaf was subjected to drying treatments in petri dishes for 0.25, 0.5, 1, 1.25, 1.5, 1.75, 2, 3, and 3.5 h and the lower parts of each leaf were used as the controls. The environmental humidity ranged from 45 to 55%. Free proline content of each part of leaves was measured as described previously (Ough, 1969), which was used as an indication of dehydration degrees and thereby to determine the appropriate periods of dehydration. 2.2. RNA preparation About 100 mg of jojoba leaf was ground thoroughly in liquid nitrogen. Powdered tissues were quickly transferred into an eppendorf tube and dissolved in 1 ml lysis buffer (2.5% SDS, 150 mM Tris–HCl, pH 7.5, and 0.5 mM EDTA). Three hun-

dred millilitres of 3 M potassium acetate solution was added and mixed thoroughly before incubation on ice for 3 min. The mixture was then centrifuged at 13,000 rpm to remove cell debris. The supernatant was recovered and extracted with 500 ␮l of a mixture of chloroform:isoamyl alcohol (24:1). The mixture was centrifuged and the aqueous was transferred to a fresh tube for further extraction. RNA was precipitated with 500 ␮l ice-cold isopropanol and incubated on ice for a few minutes before RNA pellet was collected by centrifugation and washed twice with 75% ethanol. Crude total RNA was dissolved with 60 ␮l RNase-free double-distilled water and was then digested by DNase to avoid genomic DNA contamination. The digestion was carried out by adding 3 ␮l RNase-free DNase (1 unit/␮l, Promega) and 7 ␮l reaction buffer (400 mM Tris–HCl, pH 8.0, 100 mM MgSO4 , and 10 mM CaCl2 ). Following incubation at 37 ◦ C for 1 h, RNA was purified with a RNasy Plant Mini Kit (Qiagen). 2.3. Construction of subtracted cDNA library Equal amounts of total RNAs from nine treated leaves were mixed. cDNA synthesis and SSH were carried out using SMART PCR cDNA Synthesis Kit and PCR-Select Subtraction Kit (Clontech) according to the manufacturer’s instructions. Total RNAs from dehydration treatment and untreated leaves were reverse-transcribed, respectively, to be used as the tester and the driver. Then, single-strand (ss) cDNAs were amplified for 20 cycles by PCR in a PE9700 thermal cycler (GeneAmp). During SSH, the ss cDNA tester fractions were normalized and enriched in the first two rounds of hybridizations, and stress-regulated genes were then preferentially amplified by the nested PCR. Suppression subtractive hybridization was performed in both forward- and reverse-subtractions. The secondary amplification products of the forward-subtraction were cloned into the pGEMT Easy plasmid using a T/A cloning kit (Promega). Ligated DNA was transformed to JM109 Escherichia coli cells. Colonies were cultivated for 14 h at 37 ◦ C on LB agar plates containing ampicillin, X-gal, and IPTG for blue/white colony selection. White clones were cultured in 2× YT liquid medium (tryptone 16 g, yeast extract 10 g, and NaCl 5 g, pH 7.0) containing ampicillin for 14 h. Plasmids were purified from positive clones and used in differential screening. 2.4. Differential screening of subtracted library A Differential Screening Kit (Clontech) was used to further confirm the positive clones. Colony array was conducted on four identical Hybond-XL nylon membranes (Amersham) following the protocol provided by the kit. Subtracted probes were made from the secondary PCR products of forward- and reversesubtractions. Unsubtracted tester control cDNAs (amplified in SSH processes) of both forward- and reverse-subtractions were labeled with 32 P-dCTP (3000 Ci/mmol) by random primer labeling reactions. Probes were denatured at 95 ◦ C for 7 min and chilled on ice. Membranes were prehybridized at 42 ◦ C for 4 h in 5 ml hybridization solution (50% formamide, 50 mM Hepes, pH 6.8, 2 mM EDTA, pH 8.0, 5× Denhardt solution, 1% SDS,

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and 100 ␮l/ml of salmon sperm DNA), in addition to 50 ␮l 20× SSC and 50 ␮l blocking solution, and then hybridized in the same solution at 42 ◦ C overnight in four separate hybridization tubes. The membranes were washed for 4× in 2× SSC and 0.1% SDS at 68 ◦ C and twice in 0.1× SSC and 0.1% SDS. The true positive clones were viewed with a PhosphorImager (Amersham-Pharmacia) after a 2-day exposure. 2.5. Sequence analysis Plasmid DNAs were used as the sequence templates for sequencing of inserted DNA. Sequencing PCRs were performed using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with the T7 promoter sequence primer (5 TAATACGACTCACTATAGGG-3 ). DNA amplifications were carried out in a PTC-225 DNA thermal cycler (Bio-Rad Laboratories Inc.) with the following parameters: 96 ◦ C for 2 min; 35 cycles at 96 ◦ C for 10 s, 50 ◦ C for 15 s, and 60 ◦ C for 4 min. The labeled templates were sequenced using ABI PRISM@ 3700 DNA Analyzer (Applied Biosystems). Chromatograms were analyzed by Contig Express (Invitrogen) as follows: exclude the low quality chromatograms that sequences contains many Ns, discard the sequences less than 100 bp, trim ambiguous bases at 3 and 5 end, and assemble overlapping ESTs into consensus sequences. All the clustered sequences have been documented in NCBI/DDBJ/EMBL with accession nos. from DV752668 to DV753052. For similarity search against NCBI database (http://www.ncbi.nlm.nih.gov), homologies that showed identities over 60% and e-values of less than 1e 0.5 with more than 100 nucleotides were considered to be significant. When the KEGG was used for automatic annotation, the whole gene sets were used as background with bi-directional best hit (BBH) as the assignment method. 3. Results 3.1. Proline fluctuation during dehydration treatment The extent of dehydration treatment was determined by measuring free proline content fluctuations in mesophyll cells (Fig. 1). From 0.5 to 3.5 h following treatment, proline contents of the leaves increased steadily, reached the maximum at 1.5 h, and then started to decline after 3 h. At 3 h, proline content was only 15 ␮g/g fresh leaves, and at 3.5 h the value was below 0. This result was observed in three independent experiments. Based on the proline content differences, the waterstress-regulated genes might exist with high abundance in tester mesophyll cells with concomitance wound response genes at 1.5 h following dehydration treatment, which was used for constructing the subtractive hybridization library. 3.2. Differential screening of subtracted cDNA library Although the library mainly contained differentially expressed genes, some common sequences also occurred. A typical result of differential screening experiment is shown in

Fig. 1. Proline fluctuations during water-loss (drying) treatment. X-axis represents the time points of dehydration treatment. Y-axis (proline differential content) represents the values of free proline content in treated part minus the content in non-treated part of each leaf. The value of 0 h is set as control. The leaves were regarded to be suffering from wound–water stress when their proline contents were significantly higher than the control value, and the induced genes were thought to be expressed in a higher level. Each group of treatments consists of three to nine replicates. At the time 1.5 h, proline content difference is maximum. Bars are mean ± S.D. of each group.

Fig. 2. True positive clones were selected according to the criteria described in the kit. The positive clones were hybridized to the forward-subtracted and unsubtracted tester probes but not to the reverse-subtracted and unsubtracted probes. As a result, Table 1 ESTs assembled with more than four fragments. ESTs are annotated by similarity searching against NCBI database GenBank accession no.

Annotation

Length (bp)

DV752776 DV752790 DV752788 DV752712 DV752762 DV752687 DV752752 DV752779 DV752759 DV752783 DV752732 DV752763 DV752755 DV752769 DV752773 DV752784 DV752768 DV752765 DV752785 DV752771 DV752786 DV752675 DV752684 DV752748 DV752691 DV752766 DV752725

– – – Lipid transfer protein – – GLP3 protein Metallothionein Phi-1 – Hypothetical protein – Chloroplast hypothetical protein Non-intrinsic ABC protein NtpII10 – At4g40042 – Protein of photosystem II UDP-glucuronic acid epimerase 1 OSJNBa0086P08.12 – – – – Protein of photosystem II –

673 603 799 615 308 232 808 328 567 431 837 384 553 540 525 446 517 403 590 538 213 406 525 441 693 382 277

No. of fragments 25 25 24 21 20 12 9 8 8 8 6 6 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4

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Fig. 2. Representative differential screening results. Panel a: dot blots hybridized with cDNA probes made from forward-subtracted cDNA and reverse-subtracted cDNA. Panel b: dot blots hybridized with unsubtracted cDNA probe made from unsubtracted tester control of forward- and reverse-subtraction. Positive clones were selected according to the criterion in PCR-Select Differential Screening Kit Users’ manual.

1344 positive clones were identified from the subtracted cDNA library, and 723 clones were excluded. 3.3. Features of ESTs generated We obtained 838 readable sequences after trimming. EST assembly analysis indicated that these sequences represented 385 unique ESTs, including 126 contig sequences and 259 singletons. The length of the ESTs ranged from 100 to 916 bp. Some contigs were highly repeated from 4 up to 25 fragments (Table 1), indicating that they represented highly abundant genes regulated by water deficit or related with wound stress. 3.4. Gene annotation and pathway identification A number of up-regulated ESTs associated with wound– water stress were isolated by SSH. The most worthy-studied genes need to be selected according to their roles in the pathways related to wound–water stress. Two strategies were used to find the best annotations and functional categories for these ESTs. When obtaining largescale ESTs, in general, most researchers make similarity analysis using the NCBI database, categorize them based on the data from well annotated organisms (usually A. thaliana in plants (Hara et

al., 1991)), and give a Gene Ontology (GO) (Ashburner et al., 2000) category. Gene Ontology is one of the most widely used ontologies that have been applied to annotate large number of genome databases. The GO organizes terms into three top-level categories: molecular function, biological process, and cellular component. In the case that GO terms cannot correspond directly to known pathways, a better choice is the KEGG Orthology (KO), which is a part of the KEGG suite of resources (Kanehisa et al., 2002, 2006). The KEGG gathers genes and genomes with updated annotations, and systematically analyses gene function, which aims at linking genomic information with higher order functional information. Several existing tools identify pathways in microarray data based on the KEGG pathway database, such as PathMAPA (Pan et al., 2003) and ArrayXpath (Chung et al., 2004). In this study, ESTs isolated were manually annotated by similarity blast against NCBI database. Functional classification was carried out according to the functional categories of A. thaliana proteins (http://mips.gsf.de). Out of 385 sequences, 139 sequences were annotated, which belong to 13 major categories (Table 2). The similarity information on homologous proteins mainly came from A. thaliana and 59 other plant species, including 13 woody plants and 46 herbs. KEGG Automatic Annotation System (KAAS) (http://www.genome.jp/kegg/kaas/) was then used to re-analyze all the ESTs for orthology assignment

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Table 2 Protein function catalogue annotated by homology search against NCBI database and sorted based on the protein categories of Arabidopsis thaliana proteins GenBank accession no.

GenBank match

Annotation

Organism

Identities

Metabolism Metabolism DV752764 DV752779 DV752681 DV752726 DV752737 DV752750 DV752757 DV752771 DV752781 DV752722 DV753046 DV752935 DV752928 DV752919

CAA27444 AAF68995 CAA27444 CAB88049 BAD67186 AAU05478 Q9FWK4 AAT06796 CAH60891 AAU04792 BAD94988 CAA27609 BAB84352 CAB17083

Petunia × hybrida Porteresia coarctata Petunia × hybrida Arabidopsis thaliana Phytolacca americana Arabidopsis thaliana Oryza sativa Arabidopsis thaliana Lycopersicon esculentum Fragaria × ananassa Arabidopsis thaliana Carica papaya Citrus jambhiri Hordeum vulgare subsp. vulgare

54/61 31/47 54/61 86/115 90/106 70/109 94/134 143/178 117/162 101/124 33/46 47/52 37/53 28/40

DV752916 DV752914 DV752904 DV752878 DV752874 DV752856

AAZ57445 AAL31111 AAR18402 AAQ63461 CAB77243 AAF61714

Populus deltoides Arabidopsis thaliana Nicotiana plumbaginifolia Daucus carota Persea americana Arabidopsis thaliana

54/66 60/79 116/137 148/149 102/110 61/87

DV752855 DV752824 DV752819 DV752810 DV752797 DV753006 DV752994 DV752983

ABA99594 BAD93898 AAD49420 CAI65950 CAG14979 AAC61842 AAN07898 CAA42443

Oryza sativa (japonica cultivar-group) Arabidopsis thaliana Canavalia lineata Solanum tuberosum Cicer arietinum Papaver somniferum Malus × domestica Pisum sativum

95/117 59/75 44/56 30/68 48/71 109/157 139/175 46/48

DV752958 DV752952

S22696 AAP74755

Ribulose 1,5-bisphosphate carboxylase Metallothionein Ribulose 1,5-bisphosphate carboxylase Quinone reductase-like protein Dihydroflavonol 4-reductase At1g13130 Cyanate hydratase UDP-glucuronic acid epimerase 1 Carbonic anhydrase Flavanone 3-hydroxylase Aspartyl aminopeptidase Pot. cysteine proteinase Lipoxygenase Diadenosine 5 ,5 -P1,P4-tetraphosphate hydrolase Lipoxygenase LOX2 AT5g48220/MIF21 11 Cysteine synthase Calmodulin 4 Fructose-bisphosphate aldolase 4-Diphosphocytidyl-2C-methyl-d-erythritol synthase Diaminopimelate epimerase Cyclopropyl isomerase Amine oxidase Homoserine kinase Non-cyanogenic beta-glucosidase Tyrosine/dopa decarboxylase Xyloglucan endotransglycosylase P protein; component of aminomethyltransferase Myo-inositol O-methyltransferase Chalcone synthase

Mesembryanthemum crystallinum Gypsophila paniculata

52/80 187/203

Energy DV752766 DV752773 DV752785 DV752677 DV752696

CAA59409 CAA49693 CAA59409 BAB09286 CAA78932

Spinacia oleracea Nicotiana tabacum Spinacia oleracea Arabidopsis thaliana Pinus sylvestris

59/93 107/134 52/57 68/96 108/117

DV752697 DV752731 DV752740 DV752752 DV753052 DV753042

AAO24555 AAU14832 S59550 CAA73213 AAM97921 Q04450

Arabidopsis thaliana Nicotiana tabacum Raphanus sativus Arabidopsis thaliana Chenopodium rubrum Mesembryanthemum crystallinum

72/97 52/58 62/68 130/172 51/52 46/53

DV752940 DV752906 DV752901 DV752877 DV752852

CAB88708 CAA68727 BAD05167 CAA50520 NP 565528

Spinacia oleracea Spinacia oleracea Phaseolus vulgaris Spinacia oleracea Arabidopsis thaliana

54/60 59/65 120/174 50/64 68/69

DV752836 DV753024 DV753022 DV752999 DV752953 DV752941

AAD12656 AAC21565 AAA03694 NP 564913 AAX96124 CAA45701

Prototheca wickerhamii Solanum tuberosum Mesembryanthemum crystallinum Arabidopsis thaliana Oryza sativa (japonica cultivar-group) Nicotiana tabacum

101/108 95/192 62/71 71/96 130/138 92/104

Protein of photosystem II NtpII10 Protein of photosystem II Receptor protein kinase-like protein Type 4 protein of light-harvesting complex of photosystem I At4g22300 Adenosine kinase isoform 1S H+ -transporting two-sector ATPase GLP3 protein Vacuolar proton-pumping Ppase Ribulose bisphosphate carboxylase small chain 2 PSII K-protein (UUG) ATP synthase Acid phosphatase CF(o)II ATP synthase subunit 9 Beta-hydroxyacyl-ACP dehydratase, putative H(+) -transporting ATPase, subunit 1 Lactate dehydrogenase-2a Eubisco small subunit Protein kinase family protein Adenylate kinase b (ec 2.7.4.3) 33 kDa polypeptide of water-oxidizing complex of photosystem II

142

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Table 2 (Continued ) GenBank accession no.

GenBank match

Annotation

Organism

Identities

Information pathways Transcription DV752864 DV752813 DV752808 DV752795

AAF22236 AAD22975 NP 565900 CAB88994

Pirin Cyclophilin ABC1 family protein CCR4-associated factor 1-like protein

Lycopersicon esculentum Solanum tuberosum subsp. tuberosum Arabidopsis thaliana Arabidopsis thaliana

80/92 41/45 94/219 59/87

Protein synthesis DV752774 DV752708 DV752710 DV752741 DV752742 DV752746 DV752760 DV753050 DV753039 DV752920 DV752817 DV752998 DV752985 DV752984 DV752961

AAF34765 Q40649 AAM44969 XP 478516 BAD83474 AAF26141 AAX92709 AAM92708 AAR83884 Q41365 AAB82659 BAA01974 AAC27163 AAF34800 AAD10032

60S ribosomal protein L10 60S ribosomal protein L10-3 (QM/R22). Putative 60S ribosomal protein L37 Translational initiation factor eIF1 Ribosomal protein S3 Putative 60S ribosomal protein L22 60S ribosomal protein L13a Putative ribosomal protein S18 Ly200 protein 26S protease regulatory subunit 7 Ribosome-associated protein p40 Chloroplast elongation factor TuA (EF-TuA) 30S ribosomal protein S31 60S ribosomal protein L35 Translationally controlled tumor protein

Euphorbia esula Oryza sativa Arabidopsis thaliana Oryza sativa (japonica cultivar-group) Nicotiana tabacum Arabidopsis thaliana Picea abies Triticum aestivum Capsicum annuum Spinacia oleracea (spinach) Glycine max Nicotiana sylvestris Arabidopsis thaliana Euphorbia esula Hevea brasiliensis

108/110 85/121 65/94 40/41 113/124 54/63 68/93 33/36 120/133 211/211 94/103 142/152 44/64 103/111 45/56

Silene latifolia subsp. alba

142/154

Cucumis sativus Zea mays Arabidopsis thaliana Phaseolus vulgaris Spinacia oleracea

77/97 52/71 135/171 58/61 38/40

Protein fate (folding, modification and destination) DV752789 P12332 Chlorophyll a-b binding protein, chloroplast precursor DV752718 AAQ76040 Ubiquitin extension protein DV752923 AAD28599 Bundle sheath defective protein 2 DV752877 AAM98073 AT5g24380/K16H17 9 DV752991 AAM21576 Ubiquitin-like protein SMT3 DV752960 CAA29590 Rieske FeS-precursor Protein with binding function or cofactor requirement (structural or catalytic) DV752772 7451052 Small nuclear ribonucleoprotein E homolog DV752787 CAB80522 Farnesylated protein (ATFP6) DV752701 CAA69914 Ted2 DV752704 AAT08648 ADP-ribosylation factor DV752700 AAU89264 Chloroplast light harvesting chlorophyll a/b binding protein DV753033 AAT70475 At2g26695 DV752924 CAB56693 Acyl-CoA binding protein (ACBP) DV752889 CAB96215 Profilin DV752857 AAD27878 Chlorophyll a/b binding protein CP29 DV752843 CAA98160 RAB1C DV752825 CAA96570 CP12 DV752807 CAE02442 OSJNBa0027P08.4 DV752804 CAA73720 Profilin DV752997 AAM65018 Coatomer-like protein, epsilon subunit

Medicago sativa Arabidopsis thaliana Vigna unguiculata Hyacinthus orientalis Pinus nelsonii

46/74 88/114 65/74 35/35 127/129

Arabidopsis thaliana Digitalis lanata Hevea brasiliensis Vigna radiata Lotus corniculatus var. japonicus Pisum sativum Oryza sativa (japonica cultivar-group) Mercurialis annua Arabidopsis thaliana

43/70 70/90 55/59 51/73 116/149 32/33 97/106 55/69 36/38

Protein activity regulation DV753032

Glycine max

120/175

Arabidopsis thaliana Nicotiana benthamiana Arabidopsis thaliana

56/80 146/166 54/76

Arabidopsis thaliana Atriplex nummularia Solanum tuberosum Mesembryanthemum crystallinum Arabidopsis thaliana Arabidopsis thaliana Senecio odorus Arabidopsis thaliana

123/181 74/118 144/229 156/165 130/178 83/127 50/89 37/44

BAA19608

Cysteine proteinase inhibitor

Transport Cellular transport, transport facilitation and transport routes DV752768 AAP21199 At4g40042 DV752769 AAX36074 Non-intrinsic ABC protein DV752705 AAL36253 Putative PTR2 family peptide transporter protein DV752709 AAL07001 AT4g15470/dl3775w DV752712 BAC77694 Lipid transfer protein DV752715 AAP42136 Erg-1 DV752738 AAA93521 Aquaporin DV752850 NP 565866 Glycosyltransferase family 14 protein DV752844 BAB10067 Acyltransferase DV752796 AAA33934 Lipid transfer protein. DV753013 BAC42429 Putative transport protein subunit

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143

Table 2 (Continued ) GenBank accession no.

GenBank match

Annotation

Perception and response to stimuli Cellular communication/signal transduction mechanism DV752937 BAD73736 Putative heme A:farnesyltransferase DV752858 AAB65088 AtRAB8

Organism

Identities

Oryza sativa (japonica cultivar-group) Arabidopsis thaliana

60/76 43/54

Cell rescue, defense and virulence DV753048 BAC75399 DV752802 CAC17803 DV752980 ABA00453

Manganese superoxide dismutase Peroxiredoxin Cytoplasmic Cu/Zn SOD

Nicotiana tabacum Phaseolus vulgaris Gossypium hirsutum

140/161 63/65 86/91

Interaction with the environment (systemic) DV752902 AAS98165 DV752885 AAV84588 DV752799 AAL26909

Hypersensitive-induced reaction protein Gip1-like protein Dehydration-responsive protein RD22

Capsicum annuum Populus tomentosa Prunus persica

166/195 59/71 37/42

Expansin 6

Fragaria × ananassa

75/83

OSJNBa0086O06.18 OSJNBa0086P08.12 Hypothetical protein Hypothetical protein∼predicted by FGENESH, etc. Unknown protein Hypothetical protein Chloroplast hypothetical protein Phi-1 Unknown Unknown protein At1g67350-like protein Hypothetical protein OSJNBa0014K14.1 Putative protein Type I (26 kDa) CP29 polypeptide Expressed protein Putative aldose 1-epimerase Similar to jacalin Unknown protein Unknown ORF64c Expressed protein AT5g16250/T21H19 170 Unknown protein Hypothetical protein Expressed protein Hypothetical protein

Oryza sativa (japonica cultivar-group) Oryza sativa (japonica cultivar-group) Nicotiana tabacum Oryza sativa (japonica cultivar-group)

60/93 17/46 80/81 34/34

Arabidopsis thaliana Phalaenopsis aphrodite subsp. formosana Zea mays Nicotiana tabacum Arabidopsis thaliana Arabidopsis thaliana Hyacinthus orientalis Oryza sativa (japonica cultivar-group) Oryza sativa (japonica cultivar-group) Arabidopsis thaliana Lycopersicon esculentum Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Oryza sativa (japonica cultivar-group) Arabidopsis thaliana Pinus koraiensis Arabidopsis thaliana Arabidopsis thaliana Oryza sativa (japonica cultivar-group) Oenothera elata subsp. hookeri Arabidopsis thaliana Phalaenopsis aphrodite subsp. formosana

126/157 89/91 61/72 97/136 64/125 63/83 37/53 65/80 88/100 111/129 174/187 77/91 91/112 37/65 31/35 63/90 60/68 108/140 91/128 65/99 35/47 92/153 54/61

Developmental processes Cel fate DV752930

AAK72877

Experimentally uncharacterized proteins DV752767 XP 473718 DV752786 NP 913580 DV752671 BAD83567 DV752682 NP 910689 DV752692 DV752732 DV752755 DV752759 DV752689 DV752934 DV752933 DV752917 DV752870 DV752863 DV752854 DV752847 DV752831 DV752806 DV753030 DV753026 DV753019 DV752992 DV752990 DV752974 DV752962 DV752954 DV752949

AAM13262 AAW82572 AAR91119 BAA33810 AAM62949 AAL66931 AAS20985 BAD07988 XP 473070 CAB38284 CAA43590 NP 850062 AAK64036 AAD55651 AAK21344 AAM63843 AAO74140 NP 193728 AAM74506 BAB63797 CAB67142 NP 974802 AAW82573

and pathway mapping. KO identifier confirmed 18 functional categories (Table 3). The main functional categories were carbohydrate, energy and amino acid metabolism, translation, signal transduction, and signal molecules and interaction. Although 96 genes were assigned KO numbers, 119 genes are distributed in a number of pathways, since a gene may have more than one function in different pathways. These genes were involved in 63 pathways, and the most frequent pathways are shown in Table 3. All the pathways are more or less related to water-stress response, such as glycolysis, oxidative phosphorylation, ATP synthesis, photosynthesis, carbon fixation, fatty acid metabolism, amino acid metabolism, flavonoid biosynthesis, translation and MAPK signal pathway.

Change in energy metabolism is the main response to wound–water stress in jojoba. Lipid metabolism is also an important pathway, indicating that membranes are heavily disturbed by osmotic imbalance. Translation process is especially influenced by wound–water stress since there are 12 genes involved in ribosome synthesis. Calcium signaling pathway (the secondary signaling molecules can cause receptor-mediated Ca2+ release) links the stress signal receptors and phosphoprotein cascades (Xiong and Zhu, 2001). MAPK is a component in the cascade system. In yeast and animal, MAPK pathway is responsible for production of compatible osmolytes and antioxidants. Phosphoprotein cascades regulate transcription factors, resulting in expression of stress-regulated genes.

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Table 3 Main pathways identified by KEGG Automatic Annotation System (KAAS) Pathway

Pathway no.

Genes involved

Metabolism Carbohydrate metabolism Glycolysis/gluconeogenesis Glyoxylate and dicarboxylate metabolism

ko00010 ko00630

DV752874 DV753024 DV752831 DV752797 DV753022 DV753042 DV752763

Energy metabolism Oxidative phosphorylation ATP synthesis Photosynthesis Carbon fixation Nitrogen metabolism

ko00190 ko00193 ko00195 ko00710 ko00910

DV753034 DV752699 DV752937 DV752681 DV753052 DV752906 DV752836 DV752740 DV752675 DV752940 DV752941 DV752773 DV752960 DV753022 DV753042 DV752763 DV752874 DV752757 DV752781

Lipid metabolism Fatty acid metabolism

ko00061 ko00062 ko00071

DV752939 DV752852 DV752968

Nucleotide metabolism Purine metabolism

ko00230

DV752731 DV752953

ko00272 ko00260 ko00280 ko00300 ko00340 ko00350 ko00360 ko00380 ko00400 ko00450

DV752904 DV753024 DV752983 DV752968 DV752855 DV753006 DV752914 DV752904

ko00941

DV752722 DV752737 DV753006

ko03010

DV752742 DV752870 DV753050 DV752718 DV752817 DV752927 DV752708 DV752774 DV752760 DV752746 DV753039 DV752984 DV752710 DV752792 DV752998 DV752703 DV752982 DV752802 DV752813

ko03050

DV752807 DV752920 DV753007 DV752738

ko04010 ko04020 ko04070 ko04910

DV752805 DV752912 DV752905 DV752912 DV752878 DV752878 DV752843 DV752858 DV752704

ko04060

DV752882 DV752945 DV752912

Amino acid metabolism Cysteine metabolism Other amino acid metabolism

Biosynthesis of secondary metabolites Flavonoid biosynthesis Genetic information processing Translation Ribosome

Folding, sorting and degradation Proteasome Environmental information processing Signal transduction MAPK signaling pathway Calcium signaling pathway Other signaling system Signaling molecules and interaction Cytokine–cytokine receptor interaction

4. Discussion Jojoba leaves are thick with prominent cuticle, numerous vascular bundles, no spongy layer and high percentage of dry matter, which makes the leaves mechanical rigidity and prevents wilting and enshroud visible morphological and color changes after water loss. Accordingly, it is usually hard to determine if a jojoba leaf is living or not, even if it is entirely dried for a long time. Thus, it is difficult to examine the critical water content when plant leaves wilt and to evaluate the water status of a whole living plant, since the phenotype of jojoba in response to drought or other stresses does not reflect its water status. Proline is a kind of osmoprotectant, and its accumulation is a common response of plant when suffering from water deficit (Roussos and Pontikis, 2003; Saeed et al., 2005). So proline content difference analysis is usually used for determining water status. In this study, we used detached leaves for dehydration treatment in consideration

of its convenience for treatment and minimizing errors. Based on the results from this study in jojoba, proline was accumulated in the wound–water stressed leaves at the early stage of dehydration and declined at the later stage because of decomposition. The fact of decrease of proline levels after 3.5 h below the control (the negative value) may be due to the decomposition (the activity of proline oxidase was induced highly by stress, for example) of proline in the cell or proline may be increased the decomposition after long stress. Further experiments of our study indicated that multiple genes and multiple pathways might be involved in wound–water stress responses in jojoba. The KEGG pathway database is a collection of manually drawn pathway maps based on extensive survey of published literature, allowing description of general biochemical reactions and their relationships. Based on the pathway information obtained from the KEGG, some ESTs involved in important pathways were identified. Using this approach we

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roughly summarized a strategic picture of wound–water deficit in jojoba. The main approaches for tolerance to water deficit in jojoba are osmoprotection, radical detoxification, photosynthesis protection, and energy promotion. The change in primary metabolism is a general response to stress in plants. The enzymes related to sugar metabolism are probably critical in tolerance to water stress (Leprince and Hendry, 1993). Proteins DV752874, DV752797, and DV752850 are involved in sugar metabolism. Protein changes (synthesis, degradation, and repair) were especially significant during water stress. The genes encoding proteins similar to proteases were also found in pea (Pisum sativum L.) (Guerrero et al., 1990), A. thaliana (Kiyosue et al., 1993; Koizumi et al., 1993), and maize (Zea mays L.) (Schenk and Snaar-Jagalska, 1999). A function of these enzymes is to degrade proteins that were damaged irreparably by water deficit (Guerrero et al., 1990). One strategy during early water stress is to avoid protein unfolding and to restrict membranes disturbance. During early period of drought stress in A. thaliana, mRNA encoding ubiquitin extension proteins increased in leaves (Koizumi et al., 1993). Such genes include DV752718 (ubiquitin extension protein) and DV752991 (ubiquitin-like protein SMT3). The increase may be a signal in terms of protein degradation, since ubiquitin can tag proteins for destruction. The enzymes associated with removing free radicals produced by oxygenic metabolism are important in tolerance of plants to abiotic stresses (Mittler and Zilinskas, 1994). In this study, DV752802 (peroxiredoxin) and DV753048 and DV752980 (superoxide dismutase) were induced under water stress. Loss of water and stomatal closure resulted in a reduced availability of carbon dioxide and production of active oxygen species such as superoxide radicals (Sgherri et al., 1993). Furthermore, photorespiration during drought stress was increased, which has been proved to enhance glycolate-oxidase activity and production of H2 O2 (Schenk and Snaar-Jagalska, 1999). Oxidative damage is due to the accumulation of agents such as free radicals that disrupt ultrastructure and metabolism mainly by changing the configuration of macromolecules and disruption of membranes (Farrant, 2000). Oxidative damage during water stress was thought to result from metabolic imbalances. In this study, we found that DV752928, DV752916, DV752997, DV753032, and DV752712 are involved in these metabolic processes. Another major source of oxidative damage is probably absorption of excess light energy (Smirnoff, 1993). Several genes displayed in our assay are related to photosynthesis. One explanation may be that photosynthetic electron transport is often inactivated relatively early in dehydration, while light energy continues to be absorbed by photosynthetic pigments such as carotenoids and anthocyanins (Galau et al., 1993; Giraudat et al., 1992). Some wound-regulated genes may exist in the ESTs of this SSH library, which needs to be further investigated. Understanding of the signal transduction pathways in response of plant to stresses is important in transgenic breeding of crops (Bajaj et al., 1999). The pool of ESTs provides a gene expression profile in a certain extent, which is useful in identification of certain important genes. Signal perception,

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signal transduction, and gene expression are typical responses of plants to abiotic stresses (Xiong et al., 2002). The initial reaction is to adjust stomata conductance by reducing stomatal aperture, which minimizes water loss due to inhibiting transpiration. Plant cells incline to synthesize osmoprotectants to maintain water potential (Bohnert et al., 1995). In this study, several osmoprotectants or enzymes that facilitate the synthesis of osmoprotectants were discovered. Water channel (DV752738) and channel proteins (DV752889, DV752804, and DV752923) are important in regulating water fluxes. Acknowledgements The authors thank Dr. Xiao-Yu Zhang, Zhao Yang, and Kang Liao of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China, for their assistance with differential screening and sequencing. We are grateful to Dr. Liumin Fan of Peking University, China, for his critical review of this manuscript. This work was supported by the National Natural Science Foundation of China (No. 30670203), the National Basic Research Program of China (No. 2006CB100100), and the Natural Science Foundation of Beijing (No. 5042012). References Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P., Dolinski, K., Dwight, S.S., Eppig, J.T., Harris, M.A., Hill, D.P., Issel-Tarver, L., Kasarskis, A., Lewis, S., Matese, J.C., Richardson, J.E., Ringwald, M., Rubin, G.M., Sherlock, G., 2000. Gene ontology: tool for the unification of biology. Gene Ontology Consortium. Natl. Genet. 25, 25–29. Bajaj, S., Targolli, J., Liu, L.F., David Ho, T.H., Wu, R., 1999. Transgenic approaches to increase dehydration-stress tolerance in plants. Mol. Breed. 5, 493–503. Benzioni, A., 1995. Jojoba domestication and commercialization in Israel. Isr. Hort. Rev. 17, 233–266. Benzioni, A., Dunstone, R.L., 1986. Jojoba: adaptation to environmental stress and the implication for domestication. Q. Rev. Biol. 81, 177–199. Bohnert, H.J., Nelson, D.E., Jensen, R.G., 1995. Adaptations to environmental stresses. Plant Cell 7, 1099–1111. Bray, E.A., 2002. Classification of genes differentially expressed during waterdeficit stress in Arabidopsis thaliana: an analysis using microarray and differential expression data. Ann. Bot. 89, 803–811. Chung, H.J., Kim, M., Park, C.H., Kim, J., Kim, J.H., 2004. ArrayXPath: mapping and visualizing microarray gene-expression data with integrated biological pathway resources using Scalable Vector Graphics. Nucleic Acids Res. 32, W460–W464. Diatchenko, L., Lau, Y.F., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., Siebert, P.D., 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. U.S.A. 93, 6025–6030. Farrant, J.M., 2000. A comparison of mechanisms of desiccation tolerance among three angiosperm resurrection plant species. Plant Ecol. 151, 109–115. Galau, G.A., Wang, H.Y., Hughes, D.W., 1993. Cotton Lea5 and Lea14 encode atypical late embryogenesis-abundant proteins. Plant Physiol. 101, 695–696. Giraudat, J., Hauge, B.M., Valon, C., Smalle, J., Parcy, F., Goodman, H.M., 1992. Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell 4, 1251–1261. Guerrero, F.D., Jones, J.T., Mullet, J.E., 1990. Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Mol. Biol. 15, 11–26.

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