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- Materials and Methods
- Supporting Information
The resurrection plant Craterostigma plantagineum can tolerate up to 96% loss of its relative water content and recover from such extreme dehydration within several hours (Bartels & Salamini, 2001 and Supplementary Material Fig. S1). By contrast, callus tissue of this plant has a strict requirement for exogenous abscisic acid (ABA) in order to survive severe water loss (Bartels et al., 1990 and Supplementary Material Fig. S1), and water depletion and ABA treatment result in the induction of the same set of dehydration- and ABA-responsive genes (Bartels et al., 1990). T-DNA activation tagging has allowed the isolation of callus lines tolerant to desiccation even in the absence of ABA (Furini et al., 1997; Smith-Espinoza et al., 2005). The first gene isolated from these lines, Craterostigma desiccation tolerant (CDT-1; Furini et al., 1997), when transcribed at a high level in the callus as a result of the nearby insertion of the Agrobacterium tumefaciens gene 5 promoter (pg5), induces ABA- and dehydration-responsive genes represented in this plant by the genes encoding the cDNA pc6-19, pc11-24 and pc27-45 (Bartels et al., 1990).
CDT-1 is an unusual gene: (1) the longest open reading frame (ORF) starting with ATG is followed by 20 triplets and ends with TAA; (2) no translation products have been observed; (3) no sequences comparable to that of CDT-1 have been found in databases; (4) a poly(A) tract of 17–22 nucleotides has been reported in the 5′ region of all cDNA and genomic clones, and a 3′ poly(A) tail is present in genomic clones; (5) the structure of CDT-1 is reminiscent of that of retrotransposons; it is intronless, flanked by direct repeats and present in multiple copies in the genome; (6) most importantly, CDT-1 transcript has never been detected in fresh wild-type leaves; in the callus it is up-regulated by ABA, and in wild-type vegetative organs it is induced by dehydration and repressed by rehydration (Furini et al., 1997).
Retrotransposons are genetic elements that transpose through reverse transcription of RNA intermediates; in plants they play a significant role in genome structure, organization and evolution (Kumar & Bennetzen, 1999; Brosius, 2003). The process of retrotransposition is highly invasive: retrotransposons do not excise from the genome and their copy number increases exponentially with transposition (Beguiristain et al., 2001). Plant genomes have mechanisms to control retrotransposition which limit the dangerous effects of new insertions, as well as excessive growth of the retrotransposon population (Liu & Wendel, 2000). Plant retrotransposons quiescent during normal plant development may respond to environmental stress, tissue culture conditions and pathogen attack (Kalendar et al., 2000; Beguiristain et al., 2001). Active retrotransposons can affect transcription of adjacent genes: for wheat insertion sequence Wis 2-1A, a wheat Long-Terminal-Repeats (LTRs)-retroelement, a direct link exists between retrotransposon transcription, readout activity from the LTRs and silencing or activation of the adjacent genes by means of antisense or sense RNA, respectively (Kashkush et al., 2003).
Transcriptional interference is also a mechanism by which retrotransposons might affect gene expression (Whitelaw & Martin, 2001). In plants, the notion that short interfering RNAs (siRNAs) can be essential molecules for retrotransposon regulatory mechanisms is not new. The presence of siRNAs was investigated in three different plant retrotransposons (Hamilton et al., 2002): the tobacco (Nicotiana tabacum) LTR-retrotransposon transposon Nicotiana tabacum (Tnt1) and two short interspersed elements (SINEs), TS SINE of tobacco and AtSN1 of Arabidopsis. Small RNAs of both sense and antisense polarities were detected in equal abundance. Based on the size of these siRNAs, the authors suggest that retroelement siRNAs are produced by mechanisms similar, but not identical, to those involved in transgene RNA silencing. Because retrotransposons are often transcriptionally silent, it has been suggested that siRNAs play a role in transcription inactivation (Hamilton et al., 2002). Akin to DNA transcription into active siRNA (Mallory & Vaucheret, 2006), SINE-like DNA sequences could be first transcribed into single-stranded RNA (ssRNA) and then converted into functional double-stranded siRNA. In plants, this would lead to the formation of transacting (ta) siRNA (Dunoyer et al., 2005; Yoshikawa et al., 2005).
In this study we investigated the role of CDT-1 in the pathway for desiccation tolerance. First we provide evidence that CDT-1 translation is not required, and then we show that an siRNA is responsible for opening the ABA and desiccation tolerance pathways. We lastly suggest that the ability to survive extreme conditions may have been stimulated by the environment acting on intra-genomic duplication of DNA elements.
- Top of page
- Materials and Methods
- Supporting Information
Retrotransposons are the most abundant class of transposable elements in plants (Feschotte et al., 2002). They are often considered as inert components of the genome, but their presence in databases of expressed sequence tags (Vicient et al., 2001; Nigumann et al., 2002) indicates that many have retained the capacity to initiate transcription. Moreover, the abundance of retrotransposons in most eukaryotic genomes (Lander et al., 2001; Feschotte et al., 2002), together with data indicating that retrotransposons are induced by various signals (Mhiri et al., 1997; Takeda et al., 1999), supports the idea of retrotransposons as controlling elements (Kashkush et al., 2003). It has also been reported that pseudogenes which do not produce functional full-length proteins are actively transcribed and regulate the expression of other related genes by mechanisms based on the functional significance of noncoding RNAs (Korneev et al., 1999; Hirotsune et al., 2003). The results of this study indicate that translation of the retroelement CDT-1 is not required for desiccation tolerance of the callus. However, the transcript of the 3′-end sequence of CDT-1 deleted in construct c (Fig. 1c) contains information necessary for desiccation tolerance. Another retroelement, CDT-2, isolated from a new T-DNA-generated desiccation-tolerant mutant, differs in sequence from CDT-1, but the highest sequence similarity between them is found in the region deleted in construct c. Furthermore, the CDT-2 sequence does not include the 5′ region of CDT-1 that may potentially code for a short peptide (Smith-Espinoza et al., 2005). In addition to being desiccation tolerant, transgenic calli overexpressing CDT-1 have a reddish color similar to wild-type ABA-treated calli, reinforcing the observation that a common response is elicited by CDT-1 overexpression and ABA. The obvious link between the two responses is that CDT-1 itself is ABA inducible (Furini et al., 1997). In fact, unlike vegetative organs of C. plantagineum, wild-type calli are not desiccation tolerant (Bartels et al., 1990). However, when they are treated with ABA, or transformed with CDT-1, they transcribe dehydration-related genes and become desiccation tolerant.
The analysis of the Arabidopsis genome indicates antisense RNA expression of c. 20% of the pseudogenes annotated as not-expressed sequences (Yamada et al., 2003). Moreover, transposable elements have the potential to generate double-stranded RNA as a result of promoters in their terminal repeats and/or because of their random integration in the genome (Raizada et al., 2001). In our work, in situ hybridization revealed sense and antisense RNA signals, and RNA blot analysis confirmed that both strands of CDT-1 are transcribed. Low-molecular-weight RNA analysis and the activation of the desiccation and ABA pathway in protoplasts transfected with a double-stranded siRNA identified in the 488-bp region deleted in construct c indicate that the siRNA resulting from CDT-1 transcription activates a desiccation-related response. We can speculate that the siRNA is processed from a longer double-stranded RNA precursor and represses gene expression through sequence-specific interactions with sequences of the C. plantagineum genome. The abundance of CDT-1 transcripts induced by ABA treatment, by dehydration in differentiated tissues and by the CDT-1 transgene in the callus may be recognized by the cell as a signal of stress and, through the formation of double-stranded RNA, the transcripts may be converted to small RNAs which in turn might control the expression of genes responsible for desiccation tolerance to avoid deleterious stress effects on the cell. Because in C. plantagineum only a limited number of genes implicated in the revival of dried tissues have been identified, with respect to the complexity of the desiccation tolerance phenomenon, the gene(s) potentially recognized by CDT-1 siRNA is as yet unknown. The fact that CDT-1 siRNA was detected in transgenic callus as well as in ABA-treated wild-type callus suggests an identical role for CDT-1 in both types of tissue.
Many of the newly identified tiny RNAs are not expressed at distinct stages of development but only in particular cell types (Bartels, 2004). In the callus and in planta, CDT-1 expression is restricted to special cells or cell layers (in the case of the root); however, all cells from both the callus and the root are capable of resurrecting, making it likely that the effect of the CDT-1 gene or gene products can be transferred between cells or between organs. In plants, it is known that systemic signals with RNA components (Voinnet & Baulcombe, 1997; Voinnet et al., 1998) convey RNA interference between cells. One component of this process is vascular dependent and transmits silencing signals between distinct organs. A second type of movement involves cell-to-cell contact via plasmodesmata and is based on reiterated short-distance signaling events involving 21-nt siRNA (Himber et al., 2003; Dunoyer et al., 2005). In specific cases, plant micro RNAs (miRNAs) or siRNAs are active when added exogenously to cultured plant cells (Vanitharani et al., 2003).
What is special in the case of CDT-1 siRNA is the functional link provided by the association of C. plantagineum retroelements with the increase in the level of their siRNA transcription. In establishing a mechanism capable of mediating between environment and DNA, the evolutionary relevance of the transposition of siRNA-carrying retroelements can hardly be assessed directly, but can be conjectured. The starting point is the consideration that CDT-1 mRNA accumulates in wild-type plants only during dehydration (Furini et al., 1997). This implies that the environment controls both the level of transcription of retroelements with potential siRNA activity and their genomic reinsertion. Plants do not have a sequestered germline: in meristematic cells new insertions of the element should contribute to increased desiccation tolerance in the progeny of the plant, provided that they can be transcribed under stress.
CDT-1 cDNAs vary only in the length of the 5′ oligo(A) sequence. Based on available data, a minimum of five CDT-1 elements are transcribed. This evidence was difficult to obtain because of problems with PCR amplification of genomic or transcribed CDT-1 elements across the region containing the 5′ A tract. Nevertheless, the four possible lengths of the 5′ oligo(A) sequence in CDT-1 cDNA (17, 18, 20 or 21 bp), together with the longest CDT-1 cDNA sequence already reported (Furini et al., 1997 and Supplementary Material Fig. S3), indicate that at least five CDT-1 genes are transcribed. However, a larger number is expected, based on the high frequency of CDT-1 elements in the genome, and the quasi invariance of their sequence as evident from the data on genomic clones. In sequenced cDNA clones the length of the 5′ poly(A) tract was 17, 18, 20, or 21 bp; the same tract had a length of 19, 21 and 22 bp in three different genomic clones. The multiplicity of CDT-1 transcripts is also supported by the mRNA analyses of dehydrated and rehydrated plants: a limited but clear size variability of CDT-1 transcripts has been observed (Furini et al., 1997). The sequence of genomic clones indicates that the CDT-1 genes are flanked by different DNA sequences. This, together with the high level of reiteration of the element in the genome, supports the conclusion that CDT-1 elements, after transcription under stress, are variably reintegrated in the C. plantagineum genome by retrotransposition. This is reinforced by the analysis of 10 such loci, which are all flanked by direct repeats of 5–22 bp. Furthermore, the presence of direct repeat core sequences and the different lengths of the 3′ poly(A) tails allowed, at least in part, reconstruction of the temporal series of transpositions that have increased the CDT-1 putative siRNA level. We underline the almost complete invariance of the CDT-1 sequence in the genomic clones so far considered. This is an unusual finding for plant transposons (Kumar & Bennetzen, 1999) – as if selection acted to preserve the integrity of the siRNA motif and/or of the DNA information necessary for element retrotranscription, or for double-stranded RNA synthesis. Sequence invariance supports the acquisition of adaptive traits during evolution: this is a possibility suggested for miRNA-related gene control mechanisms (Voinnet, 2004). Moreover, for the CDT-1 family, acquisition is likely to have been rapid, given the absence in plant databases of transcribed CDT-1 relatives.
To conclude, the mechanism described here provides an evolutionary explanation of the interaction between environment and DNA: the higher the expression of the retrotransposon under dehydration, the more frequent its reinsertion into the genome. As a result, there is an increasing probability that the CDT-1 element will land in a DNA sequence capable of directing transcription under stress. When repeated, such a process should result, even without selection, in a progressively higher level of synthesis of the CDT-1 siRNA, which eventually triggers the onset of desiccation tolerance. In this respect, it is worth recalling the two components of Lamarck's model of organic change (Burkhardt, 1995): the ‘power of life’, or the natural progress of organic development, can be seen as the ‘cause that tends to make organization increasingly complex’; and the modification of this progress by constraining circumstances suggests that ‘they arise from the influence of the climate, variations in temperature of the atmosphere, ... from diversities of places, ... from habits of movements’ (Lamarck, 1801, 1802).