ALSV Vector Substantially Shortens Generation Time of Horticultural Plants ALSV Vector Substantially Shortens Generation Time of Horticultural Plants

Flowering of plants is tightly regulated by both plant maturity and seasons in the year. Now that the Flowering Locus T ( FT ) gene has been revealed to encode the flowering hormone florigen, researchers are seeking to regulate and modify flowering behav - iours 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 trans ferred 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 horticul tural plants are approximately halved, and the generation time of apple plants is substan - tially 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.


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 shortday, 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, daylength 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 daylength 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 'Flowering locus T' (FT). Initially, plant biologists aimed to express the FT gene in crops by transformation, which actually accelerated flowering [3][4][5][6][7]. This suggested the functional use of FT in controlling flowering time, but transgenic plants are not typically applicable to crop production. Plant biologists then attempted nontransgenic expression of FT in crops. To our knowledge, there have been no scientific papers demonstrating the control of flowering time by application of FT protein, FT DNA or FT RNA without genetic transformations or spontaneous mutations in plant genomic DNA. We used a virus vector derived from Apple latent spherical virus (ALSV) to deriver FT RNA and express FT protein in plant tissues. This system turned out to be very successful in promoting flowering in horticultural plants. This chapter explains in detail the characteristics of the ALSV vector, activities of FT and its related genes, and how the ALSV vector is used to promote flowering.

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][10][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 Agrobacterium, and 'Agro-inoculated' into benthamiana tobacco (Nicotiana benthamiana), which is readily infected by plant viruses. Virus particles are extracted from infected leaves; viral RNAs are extracted and introduced into horticultural plants by 'particle bombardment'. The precise structures of ASLV and ALSV vector are explained below.
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 N. benthamiana if ALSV-RNA1 and ALSV-RNA2 sequences are expressed simultaneously. Expression should not necessarily be performed by genetic transformation: faster and easier protocols for transient expression are usually adopted. Once ALSV is formed in the cells of N. benthamiana, it will regenerate and spread autonomously. Three sets of different plasmid vectors have been developed for ALSV vectors ( Table 1). pEALSR1 and pEALSR2 were the original vectors used for ALSV preparation. High concentrations of these vectors are prepared from colon bacteria (Escherichia coli) culture and rub-inoculated (inoculated with SiC, carborundum) onto the leaves of quinoa (Chenopodium quinoa) plants. The so-called agro-inoculation method was more convenient, and then new vectors pBICAL1 and pBICAL2 were developed, which are typically used in our experiments. These vectors are prepared in E. coli and transformed into Agrobacterium (Agrobacterium tumefaciens). A. tumefaciens harbouring ALSV vectors are injected into the leaves of N. benthamiana for transient expression of ALSV-RNAs. pCALSR1 and pCALSR2 undergo the same procedures with pBICAL1 and pBICAL2, but appear to merit higher concentrations in E. coli. The infection rate of 'wild-type' ALSV without modification is almost 100% in N. benthamiana. ALSV vectors with exogenous insertion sequences in either cloning sites suffer from much lower infection rates in N. benthamiana. To compensate for the reduced infection rate, 'silencing suppressor' protein HC-Pro derived from other viruses (ClYVV or PVY, [22]) is also transiently expressed when ALSV-RNAs are agro-inoculated into N. benthamiana. In conclusion, triple agro-inoculation of a mixture of ALSV-RNA1 clone, ALSV-RNA2 clone and HC-Pro clone enables efficient formation of ALSV vectors.
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 SalI and MluI; Figure 2A). Another cloning site is located at the middle of the polyprotein encoded by ALSV-RNA2, between MP and Vp25 (named the XSB site, after restriction sites for XhoI, SmaI and BamHI; Figure 2B). Nucleotide sequences around the XSB site are complicated. First, nucleotide sequences around the protease-digested site (QG site) encoding 10 amino acid residues (LLEGQGPDFT) were duplicated. Second, sequences of the restriction sites were introduced

Vector
Expressed gene Backbone Antibiotics Reference Kanamycin [21] From left column to right column are shown: vector names, expressed genes, plasmid backbone origins, antibiotics for plasmid selections, and reference papers.

PVY, Potato virus Y;
ClYVV, Clover yellow vein virus. between the duplicated sequences. Finally, silent mutations (without amino acid changes) were introduced at the third nucleotides in each 10 codons surrounding the cloning site. These nucleotide modifications around the XSB site were necessary to insert exogenous nucleotide sequences at this site. The protease-digested site was duplicated such that both sites (before and after the inserted peptide) are cleaved by protease. Another special requirement for the virus vector is that the insertions are stably held by the virus. Nucleotide sequences in viruses, particularly repetitive sequences, are quite easily deleted (lost) probably via homologous recombination [ [23][24][25]. To mitigate such unfavourable reactions, mutations had been introduced around the XSB site without changing the protease-digested amino acid sequence. The third cloning site is located immediately after the stop codon of the polyprotein encoded by ALSV-RNA2 (named the MN site, after the restriction sites of MluI and NcoI; Figure 2C). These cloning sites were constructed by Li C, based on pEALSR vectors, and then copied to the other vectors.
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 Phytoene desaturase (PDS) gene. Figure 3 shows an N. benthamiana plant whose PDS gene was silenced by an ALSV vector. PDS catalyses the synthesis of carotenoids. Plants silenced in PDS expression become white, because they cannot accumulate both carotenoids and chlorophylls. This phenomenon (photo-bleaching) is believed to be caused by rapid 'photo-oxidation' of chlorophylls in the absence of photo-protective carotenoids in plant cells [28]. The representative mechanism of photo-protection by carotenoid would be non-photochemical quenching (NPQ) catalysed by PsbS protein and xanthophyll cycle. As Arabidopsis and rice mutants deficient in NPQ do not develop such white leaves caused by PDS silencing [29,30], another major and unidentified photo-protective mechanism will be exerted by carotenoids in plant cells.
Based on the degree of silencing of the apple RubisCO small subunit (rbcS) gene with the same 201-base insertion sequence, silencing effects of the three cloning sites differ from one another, and they are greater in the following order: the XSB site > the MN site > the SM site [31]. Again, insertion of a 201-base fragment of the target gene in the XSB site of the ALSV vector is recommended for efficient gene silencing with a lower risk of deletions. The SM site and the MN site are also available to silence additional target genes, by inserting different sequences at each cloning sites. Such simultaneous expression/suppression of different genes is possible, but such vectors typically show low infection rates. The MN site was also used for virus-induced transcriptional gene silencing (VITGS) in N. benthamiana and petunia, where upstream sequences (promoters) of the target genes are methylated by virus vectors [20]. The nucleotide sequence of an apple gene promoter was also mutated and inserted at the XSB site for infection to apple plants [15].

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 FT gene is grafted onto an ft mutant lacking FT gene function [32]. FT proteins produced in leaves and stems can contribute to early flowering, as well as FT proteins produced at shoot apices where flowers are formed. FT protein is originally produced in companion cells of the leaf vasculature. Then, the ALSV vector was prepared so that it expresses Arabidopsis FT gene (AtFT) at the XSB site ( Figure 4A).
Horticultural plants, such as soybean and Eustoma infected by the ALSV vector expressing AtFT generate flowers earlier than normal (Figure 4B and C) [33,34]. Such early-flowering plants often set seeds that germinate normally to generate next-generation seedlings. The ALSV vector has therefore successfully shortened the generation times of horticultural plants. Additional example of early-flowering plants is shown in Figure 5.

Differential activities of FT genes in the ALSV vector
FT genes derived from various plant species are expressed by ALSV vectors in Arabidopsis, tobacco and soybean ( Figure 6A) [11]. These FT genes are designated AtFT and AtTSF (derived from Arabidopsis), VvFT (grapevine), GtFT1 and GtFT2 (Japanese gentian), InFT1 and InFT2 (morning glory), CsFT (cucumber), PmFT (Japanese apricot), GmFT2a and GmFT5a (soybean), LeFT (tomato), CiFT (unshu mikan/satsuma mandarin), PsFTa1 and PsFTc (pea), MdFT1 and MdFT2 (apple), ComFT2 (squash) and PtFT1 (aspen). Sizes of these FT proteins range from 172 to 184 amino acid residues, but their sizes have no correlation with their activities. A previous report described the degrees of inductions of Arabidopsis and tobacco flowering by these ALSV vectors [11]. Figure 6B summarizes these data, together with unpublished data on the induction of soybean flowering.
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 FT genes, whereas, the number of leaves was more than 15 when plants were infected by the ALSV vectors expressing the remaining 9 FT genes. Among the 19 FT genes examined, AtFT and VvFT most strongly induced flowering, whereas GtFT2 and MdFT1 scarcely induced flowering. What determines the differences in the degree of flowering induction by these FT genes? Figure 6C shows the phylogenetic tree of the 19 FT proteins. The five FT proteins most strongly inducing flowering (indicated by circles) and the five FT proteins most weakly inducing flowering (indicated by triangles) are evenly distributed within the tree. This suggests that the activities of FT proteins were modified after species differentiation, rather than that their activities were evolutionarily fixed before species   differentiation. Even the MdFT1 gene, the weakest FT gene in this analysis, more or less shortened flowering time when expressed in transgenic Arabidopsis and apple [35], confirming its activity as florigen. The activities of FT genes examined in our analysis are inconsistent with grafting experiments using transgenic Arabidopsis plants ectopically expressing AtFT or AtTSF [36]. In this paper, they found that AtTSF is scarcely mobile from rootstock to scion, then fails to induce early flowering. In contrast, AtTSF was highly active when expressed by the ALSV vector in Figure 6. The activity of FT proteins as the regulator of gene expressions may matter when expressed by ALSV vectors, rather than their mobility along the phloem.
Chimeric FT genes between AtFT (strongest FT) and MdFT1 (weakest FT) were constructed and investigated in order to determine which part of the FT gene regulates its activity. This analysis was performed by Yamagishi N, and is presented for the first time here. The ALSV vectors in this analysis express chimeric FT genes at the middle of the polyprotein in RNA2 (Figure 7A). Figure 7B shows the amino acid sequences of AtFT and MdFT1. FT proteins were divided into three fragments (f1, f2 and f3); f1 corresponds to the first exon, f2 corresponds to the second and the third exons and f3 corresponds to the fourth exon of the FT genes. Chimeric FT proteins are designated C 1 FT, C 2 FT, C 3 FT, C 4 FT, C 5 FT and C 6 FT ( Figure 7C). All ALSV vectors including those expressing AtFT and MdFT1 were inoculated into N. benthamiana, but the vectors expressing C 1 FT and C 3 FT did not infect N. benthamiana. When virus-infected Arabidopsis plants were grown under a long-day condition, C 2 FT, C 6 FT, C 4 FT and AtFT strongly induced flowering, while C 5 FT and MdFT1 only weakly induced flowering ( Figure 7D). This shows that the C-terminus (f3) of the FT protein determines its strength as a florigen. Although we typically use the ALSV vector expressing AtFT to shorten generation time in horticultural plants, chimeric FT genes such as C 2 FT many more strongly induce flowering in some plant species.  [38]. The subgroup CEN is represented by the snapdragon AmCEN gene. This subgroup does not regulate flowering time, but does regulate the architecture of inflorescence (flower clusters at shoot apices). Snapdragon usually generates 'indeterminate' inflorescence, where many flowers are repeatedly generated on the side of the inflorescence, and the inflorescence continues to elongate without generating a flower at the very apex of the inflorescence. On the other hand, a snapdragon mutant in the AmCEN gene has 'determinate' inflorescence, where a limited number of flowers are generated on the side of the inflorescence, and the inflorescence stops elongating with a flower generated at the very apex of the inflorescence (called terminal flower). Thus, CEN negatively regulates flowering at the apex of the inflorescence to generate indeterminate inflorescence [39].

FT/TFL1 gene family
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. MdFT1 and MdFT2 are both expressed in apple plants. MdFT1 is suggested to regulate the flowering time of apple plants [35], but direct evidence has not yet been found. Expression levels of MdCENa are much greater than those of MdCENb [40]. Considering its classification into the CEN subgroup and high expression levels, MdCENa may regulate the inflorescence architecture of apple plants. Thus, apple plants generate clustered flowers at the apices of newly developing branches. The size of the flower clusters may be smaller in the apple mutants of the MdCENa gene. MdTFL1-1 and MdTFL1-2 share similar expression patterns. Their expression is reduced when floral buds are initiated in July [40]. According to the observations explained in the next section, MdTFL1-1 regulates the flowering time of apple plants.

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 FT genes such as AtFT with ALSV vectors (Figures 4-6). AtFT induces the early flowering of apple plants at a rate of 30% when expressed by the ALSV vector, but MdFT1 does not induce early flowering in any apple plant [10]. This result reconfirms that highly active FT homologs should be expressed by ALSV vectors to achieve early flowering, rather than endogenous but weakly active FT homologs. Consistent with this idea, other highly active FT homologs (AtTSF and GtFT1) also induced early flowering in apple plants [11]. Unlike herbaceous plants such as tobacco and soybean, the early-flowering rate of apple plants was as low as 30%, even with the most highly active FT genes. This difference between plant species may be related to their natural intervals from germination to flowering (months in herbs and years in trees).
Early flowering of plants may be also achieved through suppression of the TFL1 gene, the negative regulator of flowering. MdTFL1-1 was silenced in apple plants with ALSV vector [13]. Early flowering was observed, but in only 10% of the infected apple plants. To further improve the early-flowering rate of apple plants, simultaneous expression of AtFT and suppression of MdTFL1 homologs were performed [11]. The early-flowering rate was not increased when MdTFL1-2 was suppressed simultaneously with the expression of AtFT, but the earlyflowering rate increased to 90% when MdTFL1-1 was suppressed simultaneously with the expression of AtFT. Thus, early flowering of apple plants was successful at high rates with the combination between AtFT expression and MdTFL1-1 suppression. As well as early flowering at high rates, part of the early-flowering apple plants obtained by AtFT expression/MdTFL1-1 suppression continuously generates flowers on branches, whereas the early-flowering apple plants obtained by only AtFT expression generate flowers only once. This difference in flowering traits may be also caused by negative regulation of flowering by MdTFL1-1 in young apple plants. An example of early-flowering apple plants obtained by simultaneous AtFT expression and MdTFL1-1 suppression is shown in Figure 9. Early-flowering apple plants set fruits and seeds after pollination with the pollen gathered from other compatible cultivars. Apple fruits typically mature at about 6 months after flowering. The next-generation seedlings germinated within 1 year, counting from the germination of the mother plant [11].

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 FT genes (such as AtFT) and weakly active FT genes (such as MdFT1) according to our experiments. The analysis of chimeric FT genes in ALSV vector indicated that the difference in the activities of AtFT and MdFT1 is determined by the C terminus, rather than the N terminus. Both types of FT genes will have ecological advantages in specific plants species, but highly active types of FTs are useful for early flowering of horticultural plants with ALSV vectors. In addition, simultaneous expression of FT and suppression of TFL1 is beneficial for high rate of early flowering and continuous flowering. ALSV is not transferred to most of the next-generation plants, so the next-generation plants are neither transgenic nor infected by ALSV. Therefore, ALSV can be used as a new plant breeding technique (NPBT).