List of plasmid vectors.
Abstract
Flowering of plants is tightly regulated by both plant maturity and seasons in the year. Now that the Flowering LocusT (FT) gene has been revealed to encode the flowering hormone florigen, researchers are seeking to regulate and modify flowering behaviours by using florigen as a genetic tool. In place of transgenic approaches, Apple latent spherical virus (ALSV) vector was successful in promoting flowering of both model plants (Arabidopsis and tobacco), and fruit trees (e.g. apple, pear, and loquat), vegetables (e.g. tomato and cucumber), legumes (e.g. soybean), and ornamental flowers (e.g. petunia, Japanese gentian and Eustoma). In so doing, FT was expressed and/or TFL1 was suppressed by the ALSV vector. ALSV is a latent (non-pathogenic) virus isolated from an apple tree. After induction of flowering and seed production in crops, ALSV is not transferred to most of the next-generation seedlings, or it can be artificially removed from the infected plant by incubation at high temperature. Thus, the generation times of horticultural plants are approximately halved, and the generation time of apple plants is substantially shortened to within one year. Hence, ALSV technology is expected to be useful as a part of New Plant Breeding Techniques (NPBT) for agricultural application.
Keywords
- early flowering
- florigen
- FT
- generation time
- virus vector
1. Introduction
Breeding of horticultural plants can take several years or more. Plant cultivars are most frequently generated by crossing between different cultivars to combine various advantageous traits together, such as fruit/flower quality, pest tolerance and vigorous growth habit. Cultivars are often crossed with each other repeatedly to generate and fix such favourable traits. One generation time (from germination, flowering, seed set and germination of the next-generation seedling) is usually several months in herbaceous plants and several years in trees under field conditions [1]. Thus, crossing of herbaceous vegetables and ornamental flowers can be performed every year, while breeding of fruit trees may only be performed once every several years. If flowering could be accelerated in horticultural plants, we would be able to cross vegetables and flowers many times each year and cross fruit trees every year.
In order to cross vegetables several times in a year, problems related to the seasonality and day-length sensitivity of plant flowering also need to be addressed. Plants are either short-day, long-day or day-neutral (an aspect of the photoperiodism, the response to day length). Short-day plants flower in autumn and long-day plants flower in spring. Day-neutral plants flower in any growing season. Day length is now controlled by artificial lighting, but biotechnology to accelerate flowering will also solve the problem of day-length sensitivity without the need for regulating day length using lighting equipment. Alternatively, day-length sensitivity is also one of the important traits of horticultural plants. Ornamental flowers are harvested only when the plants set flowers. Fruits can be harvested only after the flowering seasons. Because of stable responses of horticultural plants to day length, farmers make great efforts to control time of flowering. Such artificial controls are possible only with lighting equipment, but the majority of crops are produced using sunlight alone. Gene modification by biotechnology such that a series of cultivars with different day-length responses are prepared for major horticultural cultivars will thus benefit agricultural production.
Basic studies using model plants have revealed the genes controlling flowering time in higher plants. After a functional gene is isolated in model plants, that gene and its homologs are often found to exert the same function in other plant species. This means that the flowering of vegetables, ornamental flowers and fruit trees can be controlled by this gene [2]. The name of the gene controlling plant flowering is ‘
2. Characteristics of ALSV vector
ALSV is a rare, naturally occurring plant virus isolated from an apple tree. ALSV has only been detected in a single apple tree, excluding experimentally infected plants. The infected tree was grown in the orchard of a Japanese fruit tree research institute (Morioka, Iwate, Japan). The cultivar of this apple is ‘Indo’, named after Indiana State in the United States of America. The Indo apple used to be popular in Japan, but has now been largely replaced with other cultivars such as Fuji and Orin. ALSV does not naturally spread to nearby apple trees in the orchard [8], but it can infect the seedlings of apple cultivars in the experiments [9–11]. Moreover, ALSV is a ‘latent’ virus. This means that apple plants (and fruits) infected by ALSV do not show any discernible changes, such as yellow spots, inferior growths or twisted leaves. Thus, ALSV does not induce disease, but such latent characteristics are advantageous for plant biotechnology. With the ALSV vector, we can specifically upregulate (activate) or downregulate (inactivate) functions of target genes, for example, among the 57,000 genes in the apple genome [12], with the ALSV vector. There remain other advantages of the ALSV vector. ALSV evenly infects plant tissues and then enables even silencing (inactivation) of apple genes [11, 13]. ALSV can also infect other plants and upregulate/downregulate specific genes. For instance, ALSV can infect to soybean, petunia, pea, cucumber, pear, rose, Eustoma and Japanese gentian. ALSV is latent in many plant species [14, 15]. Unlike genetic transformation, whose infection rates greatly differ between cultivars of the same crop, ALSV does not typically select cultivars. Exceptions are tomato and almond, where ALSV can infect only part of the cultivars.
The protocol for preparation and infection of the ALSV vector is precisely described in our recent publication [15]. Briefly, plasmid DNAs for expression of ALSV RNAs are prepared by ordinary procedures, introduced into
ALSV consists of two genomic RNAs: ALSV-RNA1 and ALSV-RNA2. Figure 1 shows the whole sequences of ALSV-RNA1 and ALSV-RNA2. The lengths of ALSV-RNA1 and ALSV-RNA2 are 6812 bases and 3384 bases, respectively. Similar to genomic RNAs of other plant viruses [16], genomic RNAs of ALSV encode single polyproteins. Polyproteins are translated as fused proteins and are digested into individual units by protease. ALSV-RNA1 encodes enzymes such as protease, helicase and RNA polymerase. ALSV-RNA2 encodes a movement protein and capsid proteins. The cleavage sites of the polyprotein were confirmed for ALSV-RNA2 by peptide sequences, whereas the cleavage sites of the polyprotein encoded by ALSV-RNA1 were just deduced from the peptide sequence [17]. The movement protein and the three capsid proteins are all necessary for cell-to-cell movement of ALSV within plant tissues [18].
ALSV is expected to be formed in plants such as
Vector | Expressed gene | Backbone | Antibiotics | Reference |
---|---|---|---|---|
pEALSR1 | ALSV-RNA1 | pE18PGT | Ampicillin | [9] |
pEALSR2 | ALSV-RNA2 | pE18PGT | Ampicillin | |
pBICAL1 | ALSV-RNA1 | pBICP35 | Kanamycin | [19] |
pBICAL2 | ALSV-RNA2 | pBICP35 | Kanamycin | |
pCALSR1 | ALSV-RNA1 | pCAMBIA1300 | Kanamycin | [20] |
pCALSR2 | ALSV-RNA2 | pCAMBIA1300 | Kanamycin | |
pBIN61:HC-Pro | HC-Pro (PVY) | pBIN61 | Kanamycin | |
pBE2113-HCPro | HC-Pro (ClYVV) | pBE2113 | Kanamycin | [21] |
pEALSR1 and pEALSR2 were the original vectors used for ALSV preparation. High concentrations of these vectors are prepared from colon bacteria (
It was not easy to insert exogenous nucleotide sequences into ALSV. After many trials (Li C et al., unpublished data), three cloning sites were successfully used for nucleotide insertions (Figure 2) [9, 20]. One cloning site is located immediately after the stop codon of the polyprotein encoded by ALSV-RNA1 (named the SM site, after restriction sites for
Among the three cloning sites of the ALSV vector (the SM site, the XSB site and the MN site), genes can be expressed only by their insertion into the XSB site. At the XSB site, the inserted genetic sequence is translated as a part of polyprotein, followed by digestion with protease. Cleaved proteins are expected to be attached with short peptides at both the N-terminus and the C-terminus, which derive from the protease-digested sites and the cloning site. These small attached peptides do not seem to affect the activity of inserted protein in most cases, as is experienced for transgenes introduced into transgenic plants.
All three cloning sites can be used for VIGS (virus-induced gene silencing). VIGS is a viral counterpart for gene silencing driven by antisense or inverted-repeat sequences expressed by genetic transformation [26, 27]. The virus itself is a natural target of gene silencing, then a nucleotide sequence inserted at any of the cloning sites of ALSV are silenced (degraded) by silencing mechanisms equipped in plant cells. Endogenous mRNAs harbouring the same nucleotide sequences with the inserted sequence in ALSV vector are also degraded, resulting in gene silencing. Any nucleotide sequences can be inserted at the SM site or at the MN site in principle. The nucleotide sequences inserted at the XSB site must be ‘in frame’ with the polyprotein, and this means the length of the inserts is a multiple of 3 and that each ‘codon’ encodes an amino acid without any stop codons (TAA, TAG or TGA as DNA sequences). Sizes of the inserted sequences are determined by the balance between the expected degree of silencing and potential risk of deletions (of the inserts from ALSV). Longer inserts will cause stronger silencing, but they will be more easily deleted from the ALSV vector. A length of 200 bases (or 201 bases at the XSB site) appears to be near the optimal size, with strong silencing and a relatively low possibility of deletions. Inserts shorter than 200 bases may not cause strong silencing, but the actual degree of silencing and frequency of deletions vary, depending on the inserted nucleotide sequences [14]. Insertion of nucleotide sequences at the XSB site is empirically simpler than insertions at the SM site or at the MN site. Insertions at the MN site strongly reduce the rate of viral infection, and they are also easily deleted. Insertions at the SM site have somewhat milder effects on infection rates and deletions. Insertions at the XSB site do not strongly reduce infection rate, but strongly suppress gene expression.
Model studies of silencing an endogenous gene with viral vectors often target the
Based on the degree of silencing of the apple
3. Early flowering of horticultural plants through expression of the AtFT gene
FT protein is a mobile signal of flowering. For example, FT induces flowering when a transgenic scion expressing the
Horticultural plants, such as soybean and Eustoma infected by the ALSV vector expressing
4. Differential activities of FT genes in the ALSV vector
The average number of leaves at flowering was 30 in plants infected by the control wtALSV vector. The number of leaves was more than halved (less than 15) when plants were infected by the ALSV vectors expressing 10
Chimeric
5. FT/TFL1 gene family
Among the five subgroups of FT/TFL1 gene family, FT and TFL1 regulates initiation (induction) of floral organ development and then regulates the time of flowering. FT protein is produced before the time of flowering and carried to shoot apices. As already described, FT positively regulates (increases) the expression of genes such as
As apparent in the phylogenetic tree (Figure 8), apple has two copies of each five FT/TFL1 subgroup genes. The functions of these apple genes are not completely clear, but there are several reports on their activities and expression patterns.
6. Early flowering by combination between FT expression and TFL1 suppression in apple
As already described, early flowering of horticultural plants can be achieved by expression of highly active
Early flowering of plants may be also achieved through suppression of the
7. Conclusion
ALSV will be among the most useful viral vectors for genetic engineering of horticultural plants, although ALSV is not presently applicable to cereals. Like other viral vectors, inserted sequences into the ALSV cloning sites are easily deleted upon infection to plants, depending on the sizes and sequences of the inserted sequences. What is more, ALSV vectors with insertions sometimes do not infect the plants. However, these problems can be managed by technical efforts and the selection of the insertion sequences. Nucleotide fragments larger than 1 kb can even be introduced into the XSB site of the ALSV vector and infected to plants. We hope that this chapter furthered general understanding of the structure and function of the ALSV vector, and promote its use in both basic and applied studies.
Early flowering through infection of ALSV vectors shortens the generation times of horticultural plants. This technique is expected to promote breeding of horticultural plants. It may not be a popular concept, but there seems to be strongly active
Acknowledgments
We like to acknowledge Kiyoaki Maeda, Tomiko Kawaguchi, all laboratory members and collaborators for helpful supports in virus infection to horticultural plants.
Funding
This study was supported by the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry (Japan).
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