LoMYB20s in lily belongs to the R2R3-type MYB gene family
As is well known, many MYB-family genes participate in the anther development process, in a variety of roles [20, 21]. In order to obtain MYB genes with roles in anther dehiscence in lily, we designed a degenerate primer based on the highly conserved R3 DNA-binding domain of the MYB family, and finally obtained a full-length sequence of a MYB gene from mature anthers by the RACE amplification method, which was deposited in Genbank with the designation LoMYB20s and the accession number KT759161. The full-length nucleotide sequence of LoMYB20s was 807 bp, encoding a deduced protein sequence of 190 amino acids. LoMYB20s has two MYB DNA-binding domains (Fig. 1a), and is a R2R3-type MYB protein [19]. A BLAST homology search of the sequence of LoMYB20s against the NCBI protein database revealed ten similar sequences (Fig. 1a), but no study of these MYBs has so far been reported. In Arabidopsis, the R2R3-type MYB factors that are encoded by 125 MYB genes have been categorized into 22 subgroups [19]. LoMYB20s is most closely related to MYB57, MYB21 and MYB24 from subgroup 20 of Arabidopsis, and then to MYBs from subgroup 19 (Fig. 1b).
There is only one copy of LoMYB20s in the lily genome
Southern hybridization was used to identify whether there were multiple copies or close homologs of LoMYB20s in the lily genome. The full-length sequence of LoMYB20s was used as a probe, and endonucleases BamHI, EcoRI, HindIII, and XbaI were used for genomic DNA digestion. The results showed that two bands were visible in the EcoRI-digested product, and that only one band was visible in the other three endonuclease-digested products (Fig. 2). Given that there is a single EcoRI cleavage site in the LoMYB20s sequence, the results suggested that LoMYB20s is a single-copy gene, with no close homologs in the lily genome.
LoMYB20s is mainly expressed in the late stages of lily anther development
Levels of expression of LoMYB20s were examined in a range of tissues of the lily plant, including stem, leaf, petal, ovary, stigma, filament, anther, anther wall, and pollen. LoMYB20s was found to be strongly expressed in petal, stigma, filament, anther, and anther wall, but not in leaf, stem, pistil, or pollen (Fig. 3a). Expression levels of the gene were also determined at different anther developmental stages. The various stages of anther development were easily distinguishable by anther color, which progressed from white to green, then to yellow, and finally to purple [28]. A low level of expression of LoMYB20s was detected at the white and green stages, which increased at the yellow and purple stages, and then declined following anther dehiscence (Fig. 3b). These results suggested that LoMYB20s probably functioned during the late phase of anther development.
LoMYB20s silencing inhibits anther development and dehiscence
To examine the possible role of LoMYB20s in anther dehiscence, we silenced LoMYB20s in lily using virus-induced gene silencing (VIGS) [28]. Total RNA was extracted from anthers of lily plants infected with Agrobacterium tumefaciens carrying pTRV-LoMYB20s or the pTRV empty vector and analyzed by semi-quantitative RT-PCR (Fig. 4a). LoMYB20s was successfully silenced in the anthers of six lines of lilies (6/100), and representative LoMYB20s-silenced flowers of lily are shown in Fig. 4b. We observed that, in the LoMYB20s-silenced lilies, the flower petals were well developed and opened normally, and the filaments and stigmas appeared to be normally elongated; however, the anthers became slender, and indehiscent (Fig. 4b). Conversely, in the control lines, the anther walls could roll outwards and the anthers were able to dehisce normally. These results indicated that silencing of LoMYB20s affected anther development, and demonstrated that LoMYB20s plays important roles in anther dehiscence.
Dexamethasone (DEX) induction of LoMYB20s led to senescence and infertility in Arabidopsis
To further investigate the function of LoMYB20s in anther development, we constructed the pTA7001G-LoMYB20s vector, in which LoMYB20s is overexpressed only under DEX induction, and used this vector to transform Arabidopsis thaliana. Under the control treatment (no DEX treatment), DEX::LoMYB20s transgenic lines (LoMYB20s-expressing plants), wild-type (WT) plants, and pTA7001G transgenic plants (control plants) all grew, flowered, and produced siliques normally; the only noticeable effect was that the inflorescence stems of the LoMYB20s-expressing plants were a little short compared to those of the control plants (Additional file 1: Figure S1). Under DEX treatment, the control plants grew, flowered, and bore seed normally, whereas the LoMYB20s-expressing plants displayed serious leaf yellowing within a few days (Fig. 5a) and their inflorescences were poorly developed, or developed to be procumbent and with fewer siliques (Fig. 5b). These results indicated that overexpression of LoMYB20s caused early senescence of the transgenic plants, and severely influenced inflorescence development in Arabidopsis.
MYB homologs were induced in LoMYB20s transgenic Arabidopsis
In an attempt to explain the phenotypes of LoMYB20s-silenced lily anthers and LoMYB20s-expressing Arabidopsis, the expression levels were detected of potentially related genes in Arabidopsis, using RT-PCR (Additional file 1: Figure S2); this revealed that several different types of genes were obviously induced.
In LoMYB20s-expressing Arabidopsis, the expression of LoMYB20s and its closest MYB homologues was initially examined (Fig. 6a, Additional file 1: Figure S2a). As anticipated, the expression of LoMYB20s was only detected following DEX treatment, and not in the controls, indicating that the phenotype of the transgenic plants sprayed with DEX was consequent upon LoMYB20s expression. Then, the expression of the closest homologues of LoMYB20s in Arabidopsis, such as MYB21, MYB24, and MYB57, was determined. This revealed that both MYB24 and MYB57 were induced, though expression of MYB21 was not detected. Next, the expression of various downstream genes of these homologous MYBs [20] was determined: the phenylalanine ammonialyase genes PAL1 and PAL2, the terpene synthase genes TPS11 and TPS21, the alternative oxidase gene AOX1a, and the auxin-related genes SAUR63, IAA2, IAA3, IAA7, IAA19, ARF6, and ARF8. Of these, only the expression of PAL2 and TPS21 showed clear changes.
Expression of PCD- and SWB-related genes was increased in LoMYB20s transgenic Arabidopsis
Given that the rosette leaves of the transgenic Arabidopsis seedlings showed premature yellowing, RT-PCR was first used to search for alterations in the expression of PCD-related genes (Additional file 1: Figure S2b). Several genes showed altered expression in the DEX-induced plants. Their expression was then precisely quantitated using qRT-PCR, which revealed dramatically higher levels of expression, relative to controls, in the LoMYB20s-expressing plants (Fig. 6b). These genes included, amongst others, three pathogen-elicited PCD-associated genes, PR-1, PR-2, and PR-5 [29], a KDEL-tailed serine protease (CysP) gene, CEP2 [23], a senescence-associated CysP gene, SAG12 [31], a vascular xylem autophagy modulator gene, MC9 [32, 33], and a senescence-associated TF gene, WRKY53 [34].
As indicated previously, in LoMYB20s-silenced VIGS lilies, the anther wall was unable to roll outwards and the anthers dehisced abnormally, which suggested that SWB might be disrupted. Accordingly, we examined the expression in transgenic Arabidopsis of a number of genes involved in SWB (Additional file 1: Figure S2c). Among these, three negative regulator genes, MYB7, MYB32, and KANT7/IRX11 [35, 36] , and two genes encoding proteins that interact with the KANT7 gene product, BLH6 and OFP4 [37], were obviously induced (Fig. 6c). In addition, two effector genes involved in xylan biosynthesis, FRA8/IRX7 [38] and IRX15L/DUF579 [39], and an endo-PG gene, QRT2 [5], were also sharply induced in LoMYB20s-expressing plants (Figure 6c).
Genes of JA and GA metabolism were altered in transgenic Arabidopsis
Among genes downstream of LoMYB20s that have been mentioned above, some have been reported to be JA- and/or GA-regulating genes, such as MYB24, 32, 57, WRKY53, SAG12, and QRT2. Therefore, the expression of genes involved in the metabolism and signaling of JA and GA was examined, including JA biosynthetic and metabolic genes, DAD1, LOX1-6, AOS, AOC, OPR3, ACX1, JMT, and JAR1, the JA early signal transduction genes, COI1, MYC2, MYC3, and MYC4, GA-biosynthetic genes GA20ox1-5 and GA3ox1-4, GA-deactivating genes GA2ox1-8, and DELLA genes (Additional file 1: Figure S2d). Two JA biosynthetic genes, LOX1 and JMT [40], and three genes of GA deactivation, GA2ox4, GA2ox6, and GA2ox8 [41], were induced in LoMYB20s-expressing plants compared to the control plants after DEX treatment (Fig. 6d). These results suggest that LoMYB20s could be involved in JA/GA-associated stamen developmental regulation.