CDT retroelement

Abstract

The resurrection plant Craterostigma plantagineum can tolerate up to 96% loss of its water content and recover from such extreme dehydration within several hours. This property is not shared by callus which has a strict requirement for exogenous abscissic acid (ABA) to survive severe water loss. ABA treatment and dehydration result in the induction of similar drought-responsive genes. Activation tagging led to the isolation of CDT-1 gene which renders callus desiccation tolerant bypassing the ABA requirement. This gene belongs to a retroelement family, members of which are induced by ABA and dehydration in callus, supporting its role in desiccation tolerance. Indeed, CDT genes have been detected in other desiccation tolerant Craterostigma species. CDT-1 RNA of both strands was identified by in situ hybridization and a CDT-1-derived short interfering RNA was detected in desiccation tolerant tissues and was able to induce dehydration genes in transfected protoplasts to the same extent as an ABA treatment. Thus, under environmental stress the induced transposition, over generations, directs the amplification of CDT-copy number in the genome and increases the desiccation tolerance phenomenon.

Plants have evolved different strategies such as morphological, physiological and biochemical adaptations to reduce the unfavorable effects of limited water availability.1 ,2 These strategies allow plants to survive in arid environments by lessening the severity of water stress but with long exposure to drought these plants will dehydrate and die.³ Among vascular plants a small group of angiosperms known as resurrection plants⁴ can tolerate extreme dehydration; The acquisition of desiccation tolerance in the resurrection plant Craterostigma plantagineum (Fig. 1) is mediated by abscissic acid (ABA) and is associated to the de novo synthesis of proteins and sugars, which are postulated to function as cellular protectants.⁵ Conversely, in vitro propagated callus is not desiccation tolerant and it requires exposure to ABA in order to survive severe dehydration.⁶ Treatment of callus tissue with ABA induces most of the genes that are induced by dehydration in the whole plant.

Figure 1.

Effect of desiccation treatment on the resurrection plant C. plantagineum. Fully turgid (A), desiccated (B) and rehydrated (C).

T-DNA activation tagging experiments have permitted the isolation of callus lines tolerant to desiccation in the absence of ABA.^7,8 The first gene identified from these lines, CDT-1, when transcribed at high level in callus, leads to the expression of dehydration and ABA-responsive transcripts. CDT-1 shows hallmarks of non-LTR retrotransposons: it does not encode a functional protein, it is present in multiple copies in the genome, possesses a 3′ poly(A) tail and is flanked by direct repeats.⁷ CDT-1 is induced in leaves by dehydration and suppressed as soon as water is supplied, whereas in wild-type callus it is upregulated by ABA.⁷ Transformation of Craterostigma with mutated versions of CDT-1 confirmed that CDT-1 protein is not required for callus desiccation tolerance and that the transcription of the 3′ part of the CDT-1 sequence is needed to confer desiccation tolerance on callus. RNA blot analysis indicated that CDT-1 is transcribed from both strands and in situ hybridization showed CDT-1 signal only in cells with extensive cell wall thickening of ABA-treated callus, and in a thin layer of parenchyma cells of ABA-treated roots.⁹ Furthermore, both sense and antisense 21-mer probes designed on the 3′ end of CDT-1 cDNA hybridized with low molecular weight RNA from desiccation tolerant tissues. This last observation suggested that CDT-1 mRNA can play a regulatory role in the process of desiccation tolerance through a mechanism of siRNA. Indeed, in a transient expression assay with Craterostigma protoplasts siRNA corresponding to this 21 mer on the 3′ end CDT-1 the transcribed region of the element whereas the analysis of genomic clones indicated that CDT-1 genes are flanked by different direct repeats.^7,9

Continuing with the activation tagging approach, other mutant callus lines were identified with the ability to survive dehydration without an ABA pre-treatment and, analysis of one of the tagged loci led to the identification of %CDT-2. Surprisingly, this gene shows high similarity with CDT-1, particularly, they share sequence motifs within the 3′ region.⁸ Other desiccation tolerant species of the genus Craterostigma: C. hirsutum, C. pumilum and C. lanceolatum were analyzed for the presence of CDT-1 homologs. PCR analysis with different primer combinations, all designed on CDT-1 cDNA sequence, allowed the identification of CDT-genes in the three species. In all cases sequence similarities were identified within the 3′ part of CDT-1 sequence (Fig. 2). This result confirmed that CDT-like genes are present in desiccation tolerant Craterostigma species. They are probably essential for surviving extreme dehydration and the information required for inducing desiccation tolerance is positioned in the 3′ part of the gene sequence.

Figure 2.

Alignment of full length CDT-1 sequence from C. plantagineum with portions of CDT-genes identified in C. hirsutum, C. pumilum and C. lanceolatum. Primers used for PCR amplification are identified with asterisks.

Plant retrotransposons can be inactive under normal plant development and activated by environmental stress.^10,11 The increased copy number due to their transposition will increase chances of inheritance by the next generation and hence transposon survival.¹² Retrotransposons are often considered inert components of the genome, however their abundance in most eukaryotic genomes^13,14 and their presence in databases of expressed sequence tags¹⁵ suggests that many of them have preserved the transcription capacity. In addition, it is known that transposable elements have the potential to generate double-stranded RNA.¹⁶ In the case of CDT-1 double-stranded RNA may be a precursor of siRNA interacting with the sequence of Craterostigma genome. This siRNA may work as a transacting small interfering RNA¹⁷ and direct the cleavage of a repressor, controlling the pathway that leads to desiccation tolerance, with which it may also share limited sequence similarity. Alternatively, the CDT-1 siRNA could mediate silencing by affecting the methylation status of gene(s) responsible for the expression of desiccation-and ABA-responding genes. What is singular in the case of CDT-1 retroelement is its abundant transcription in the whole plant when water stress occurs, and its reinsertion in the genome, confirmed by the high number of genomic clones identified, and by the difference in their flanking sequences. Thus, over generations, drought will increase CDT retroelements retrotransposition and the level of siRNA that opens a desiccation tolerant pathway leading to plant resurrection. It is worth emphasizing the presence of the CDT gene family in desiccation-tolerant Craterostigma species and the conservation only of the 3′ part of the element, as preservation of this part of the sequence and the interaction between environment and DNA during evolution would have ensured the continuance of the desiccation tolerant trait.

Addendum to: Hilbricht T, Varotto S, Sgaramella V, Bartels D, Salamini F, Furini A. Retrotransposons and siRNA have a role in the evolution of desiccation tolerance leading to resurrection of the plant Craterostigma plantagineum. New Phytol. 2008;179:877–887. doi: 10.1111/j.1469-8137.2008.02480.x.