JXB Advance Access originally published online on September 9, 2003
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Journal of Experimental Botany, Vol. 54, No. 392, pp. 2593-2595,
November 1, 2003
© 2003 Oxford University Press
Photosynthetic genes are differentially transcribed during the dehydration-rehydration cycle in the resurrection plant, Xerophyta humilis
Received 19 May 2003; Accepted 22 July 2003
Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, South Africa
* To whom correspondence should be addressed. Fax: +27 21 689 7573. E-mail: collett{at}science.uct.ac.za
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Abstract |
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One of the desiccation-tolerant mechanisms of the resurrection plant, Xerophyta humilis, is the ability to shut down photosynthesis reversibly. The X. humilis psbR and ChlP genes, encoding the 10 kDa polypeptide of photosystem II (PSII) and a geranylgeranyl reducatase, respectively, were isolated in a differential display screen as dehydration-down-regulated and rehydration-up-regulated transcripts. Two other PSII genes, psbA (chloroplast-encoded) and psbP (nuclear-encoded), isolated by degenerate primer PCR, display a similar trend in expression.
Key words: Dehydration–rehydration cycle, photosynthesis, photosystem II genes, resurrection plant, transcript.
Xerophyta humilis (Bak.) Dur and Schinz is a monocotyledonous southern African resurrection plant that survives dehydration to 5% relative water content (RWC). As a protective mechanism against dehydration-induced oxidative stress, poikilochlorophyllous Xerophyta species reversibly lose chlorophyll and the thylakoid membranes are dismantled into small vesicles (Sherwin and Farrant, 1996, 1998). It is speculated that this is a controlled process that allows for the reassembly of chloroplasts during rehydration. Consequently, the reassembly of the chloroplasts and restoration of photosynthetic activity are one of the major activities in the rehydration phase.
Two dehydration-down-regulated, rehydration-up-regulated cDNAs (XH13, 219 bp and XH8, 150 b) were isolated by differential display PCR using primers H-T11C (5'-AAGCTTTTTTTTTTTC-3') and H-AP3 (5'-AAGCTTTGGTCAG-3' from the RNAimageTMKit1 (GenHunter Corporation). Differential expression was confirmed by reverse northern blotting and the fragments were cloned into the PCR-TRAP vector (Genhunter Corporation) and sequenced.
The full-length cDNA product (
700 bp) for clone XH13 was isolated by 5' SMARTTM RACE PCR (Clonetech) using the gene-specific antisense primer, 5'-CAGAGTAAATACACACAGACAG TGCC-3' and cloned into pGEM-T-easy vector (Promega). BlastX analysis of the sequenced product identified it as the nuclear-encoded psbR gene (417 bp; 139 aa), specifying the 10 kDa protein of photosystem II (PSII). The amino acid sequence alignment is shown in Fig. 1A. According to Pairwise Blast analysis, the product shares 72% amino acid sequence identity and 88% similarity with the equivalent psbR polypeptide from rice.
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Based on sequence homology between the 17 aa of the 3' translated region of XH8, and the C-terminal end of the corresponding gene from tobacco (Blastp E=0.65, 70% identity, 90% similarity), XH8 was tentatively identified as ChlP, encoding a geranylgeranyl reductase. Attempts to isolate the full-length cDNA by 5' RACE PCR were unsuccessful, and so to confirm the identity of XH8, a ChlP 5' degenerate primer (5'-TGGGT(A/T/G/C)TT(C/T)CC(A/C/G/T)AA(A/G)TG(T/C)GA(CT)C-3') was used in combination with a XH8-specific primer (5'-GAGTGAGCTGCTGC ACCCGCAAC-3'). The expected 650 bp product was cloned into pGEM-T-easy vector (Promega) and sequenced. According to Pairwise Blast analysis, the truncated ChlP gene shares 56% amino acid sequence identity and 74% similarity with the rice geranylgeranyl reductase gene (452 aa). Homology with the Arabidopsis ChlP (472 aa) is significantly higher: 83% and 93% amino acid identity and similarity, respectively. Alignment with the Arabidopsis and rice genes is shown in Fig. 1B.
To compare the expression of other PSII genes in response to the dehydration–rehydration cycle, internal fragments of the nuclear-encoded psbA (825 bp), and chloroplast-encoded psbP (996 bp) were isolated from 5' RACE-ready cDNA by degenerate primer PCR using the following primers: 5'-TGAC(T/C)GCAATT(T/A)TAGA (G/A)AGACGCG-3' (forward primer) and 5'-CGTTC(A/G)TGC AT(A/T)AC(T/C)TCCATACC-3' (reverse primer) for psbA and 5'-AAGCAGTGGTAACAACGCAGAGT-3' (the 5' SMART RACE nested universal primer) and 5'-GC(T/A/G)CC(C/T)TT(G/A)AAC CA(C/T)CTCTTGTC-3' (reverse primer). The X. humilis psbA (Genbank accession no. AF545583 [GenBank] ) and psbP (AF545584 [GenBank] ) fragments share 90% and 69% amino acid sequence identity with the respective rice homologues.
Northern blot analysis (Fig. 2) shows that psbR 0.8 kb transcript levels decrease during dehydration and are up-regulated within 12 h of rehydration (40% RWC). PsbA (1.4 kb) and PsbP (1.0–1.2 kb) show a similar trend in expression. Like psbR, psbP is up-regulated within 12 h of rehydration (40% RWC), whereas psbA up-regulation occurs within 24 h of rehydration (85% RWC). This may reflect delayed transcription for chloroplast-encoded genes dependent on chloroplast reassembly. ChlP transcripts (1.6 kb) are present at low steady-state levels in the fully-hydrated plant and are not detected in dehydrating X. humilis leaves. As for psbP and psbR, ChlP transcription is strongly induced within 12 h of rehydration (40% RWC), and transcript levels accumulate in the course of the rehydration cycle. Within 72 h of rehydration, ChlP transcripts have not reached steady-state levels corresponding to those in the fully hydrated plant (100% RWC) prior to dehydration.
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Low levels of the PsbA and PsbP transcripts appear to be stably maintained in the desiccated plant. In investigating the rehydration response in X. humilis, Dace et al. (1998) have shown that the partial recovery of PSII function on rehydration is independent of transcription. This implies that the necessary transcripts are ‘stored’ for immediate translation within the first phase of rehydration. PsbA constitutes the reaction centre protein D1 and may be required for initial assembly of PSII. The function of the 10 kDa polypeptide is largely unknown, but it seems to play a structural role in providing a binding site for a 23 kDa PSII protein (PsbP) to the surface of the thylakoid membrane (Lautner et al., 1988). CHLP catalyses the reduction of geranylgeranyl diphosphate to phytyl diphosphate, providing phytol for chlorophyll and tocopherol synthesis. ChlP antisense tobacco transgenics show reduced chlorophyll and tocopherol content (Graßes et al., 2001).
Because PSII is responsible for the water-splitting, oxygen-evolving functions of photosynthesis, and drives the strongest oxidizing reaction known to occur in nature, loss of PSII function, correlates with loss of power to generate potentially damaging reactants and, therefore, PSII may act as a sensor for stress (van Rensen and Curwiel, 2000).
In contrast to this study, PsbR, PsbP and ChlP (designated RAFL05-17-F20, RAFL03-03-A07 and RAFL104-16-B07, respectively) are reported by Oono et al. (2003) as genes that are not up-regulated during rehydration in Arabidopsis.
This study provides molecular evidence that supports physiological evidence of the plant dismantling and reassembling its photosynthetic machinery during dehydration and rehydration, respectively.
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Acknowledgements |
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We thank Halford Dace for the differential display PCR screening. This work was supported by an Innovation Fund Grant (Department of Arts, Science and Technology), the National Research Foundation, and the University of Cape Town.
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TopAbstractReferences |
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Dace H, Sherwin HW, Illing N, Farrant JM. 1998. Use of metabolic inhibitors to elucidate mechanisms of recovery from desiccation stress in the resurrection plant Xerophyta humilis. Plant Growth Regulation 24, 171–177.
Graßes T, Grimm B, Koroleva O, Jahns P. 2001. Loss of 
-tocopherol in tobacco plants with decreased geranylgeranyl reductase activity does not modify photosynthesis in optimal growth conditions but increases sensitivity to light stress. Planta 213, 620–628.[CrossRef][Web of Science][Medline]
Lautner A, Klein R, Ljungberg U, Reiländer H, Bartling D, Andersson B, Reinke H, Beyreuther K, Herrmann RG. 1988. Nucleotide sequence of cDNA clones encoding the complete precursor for the ’10 kDa’ polypeptide of photosystem II from spinach. Journal of Biological Chemistry 263, 10077–10081.
Oono Y, Seki M, Nanjo T, et al. 2003. Monitoring expression profiles of Arabidopsis gene expression during rehydration process after dehydration using c. 7000 full-length cDNA microarray. The Plant Journal 34, 868–887.[CrossRef][Web of Science][Medline]
Sherwin HW, Farrant JM. 1996. Differences in rehydration of three desiccation-tolerant angiosperm species. Annals of Botany 78, 703–710.
Sherwin HW, Farrant JM. 1998. Protection mechanisms against excess light in the resurrection plants Craterostigma wilmsii and Xerophyta viscosa. Plant Growth Regulation 24, 203–210.
Van Rensen JJS, Curwiel VB. 2000. Multiple functions of photosystem II. Indian Journal of Biochemistry and Biophyics 37, 377–382.
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