The R2R3-type LoMYB20s gene regulates programmed cell death and secondary wall biosynthesis, and affects anther development and dehiscence in Lily CURRENT STATUS: POSTED

Background Lilies are the widely cultivated cut flowers worldwide, while lily anthers carry a large amount of colored pollen dispersed easily to stain petals that makes serious problems for commerical sales. Improving pollen pollution in lily is one of the major goals of lily breeding. Results In this study, we identified a putative R2R3 MYB transcription factor LoMYB20s from oriental lily ( Lilium spp. ‘Siberia’). LoMYB20s mainly expressed in anther wall during the late stages of lily anther development. Suppression of LoMYB20s by virus-induced gene silencing (VIGS) in lily led to a failure of the anthers to dehisce. Induction of LoMYB20s in DEX::LoMYB20s transgenic Arabidopsis caused the rosette leaves turning yellow and the inflorescences becoming procumbent and infertile. And the downstream genes of LoMYB20s were involved in multiple metabolic processes including jasmonate (JA) biosynthetic, gibberellin (GA)-deactivating, programmed cell death (PCD), and secondary wall biosynthesis (SWB). These results suggested that LoMYB20s participated in anther development and dehiscence possibly through regulating the PCD and SWB processes in a JA/GA-associated manner. Conclusions Our results demonstrated the indispensable role of LoMYB20s in lily anther development and dehiscence, and provide a possibility of using LoMYB20s silencing to produce anther-indehicent lilies.

Multiple types of transcriptional factors (TFs) and enzymes have been found to participate in this late stage of anther development; however, overall, the process remains largely uncharacterized [15,16,17]. R2R3-MYBs comprise one of the largest families of plant TFs and are important regulators playing diverse roles in anther development [18,19]. In Arabidopsis, MYB21, MYB24, and MYB57 are critical components in the JA-mediated transcriptional cascade for stamen development and regulate overall filament elongation and anther dehiscence [20,21]. In addition, the anther-specific gene MYB26 governs SWT in the endothecium and functions upstream of the central TF genes involved in secondary wall (SW) formation, including NAC SWT promoting factor1 (NST1) and NST2 [10,11]; the GAMYB-like genes MYB33/MYB65 facilitate tapetal and pollen development [22]; and MYB80 is required for tapetal cell death, callose dissolution, exine formation, and pollen development in Arabidopsis [23,24] and are functionally conserved in crops such as cotton, lily, and Brassica [25,26,27].
Lilies are amongst the most important and widely cultivated cut flowers worldwide; however, lily anthers carry a large amount of highly colored pollen. This easily becomes dispersed when the flower opens, leading to unsightly staining of the flowers themselves and of items that come into contact with it, such as clothing. Research into lily anther development and dehiscence will not only reveal the fundamental mechanisms involved in these processes, but will have practical implications in providing approaches for the control or prevention of pollen release in the breeding of new hybrid cultivars. In the present study, we report the functional characterization of a single R2R3-type MYB gene LoMYB20s from lily, which is specifically expressed in the late phase of anther development.
Investigation of the effect of LoMYB20s silencing on lily anther development showed an inhibition on anther dehiscence, and it might be resulted from the obstruction of PCD-related anther tissue degeneration and the disturbance of natural SWB, possibly via a JA/GA (jasmonate/gibberellin) comediated regulatory pathway.

Results
LoMYB20s in lily belongs to the R2R3-type MYB gene family 4 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 R2R3type 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.

Arabidopsis
To further investigate the function of LoMYB20s in anther development, we constructed the  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 6 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.

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.

Discussion
Regarding its homology to MYB proteins in Arabidopsis, LoMYB20s is most closely related to MYB21, MYB24, and MYB57 (Fig. 1b). MYB21, MYB24, and MYB57 are closely related to each other and they have been demonstrated to have a role in the JA regulation of pollen maturation, filament elongation, and anther dehiscence [21]. In contrast to the shorter filaments and petals displayed in the delayed dehiscence phenotypes exhibited by myb21, myb21myb24, or myb21myb24myb57 mutants [20,42], only slender and indehiscent anthers were observed in LoMYB20s-silenced lily flowers (Fig. 4b). In transgenic LoMYB20s-expressing Arabidopsis plants, MYB21 expression was only slightly influenced, compared to MYB24 and MYB57, which were strongly induced (Additional file 1: Figure S2, Fig. 6a). MYB21, MYB24, and MYB57 are the key TFs in JA-regulated anther development, but the mechanism by which mutations in MYB21, MYB24, or MYB57 cause the phenotypes mentioned above has not been elucidated. Several genes have been reported to be downstreams of MYB21 or MYB24, such as PAL, AOX1a, and TPS11 and TPS21 [42,43]. Among these genes, only PAL2 and TPS21 were significantly induced in LoMYB20s-expressing Arabidopsis plants after DEX induction (Additional file 1: Figure S2a, Fig. 6a). A number of auxin response and JA biosynthesis genes have been shown to display obvious changes in myb21myb24 double mutants, including SAUR63, IAA2, IAA3, IAA7, IAA19, DAD1, and LOX2 [42], but none of them showed significant change in LoMYB20s-expressing plants in the present work (Additional file 1: Figure S2a). It would appear that LoMYB differentially affected the expression of these MYBs and their downstream genes and that, perhaps as a consequence of this, LoMYB20s regulated anther dehiscence more specifically than its homologs.
Given that LoMYB20s-expressing Arabidopsis plants exhibit a phenotype of early senescence after DEX induction (Fig. 5a), it is reasonable to suspect that LoMYB20s participates in the regulation of PCD in lily anthers, where it is expressed. In the Arabidopsis MYB family, several MYBs have been reported to be associated with PCD, including MYB33, MYB65 [22], MYB80 [23], MYB101 [44], and MYB30 [45]. None of these genes showed changes in expression in LoMYB20s-expressing plants after DEX induction (Additional file 1: Figure S2b). But one of the downstream targets of MYB30, PR-1 [46] showed strongly altered expression in both LoMYB20s-expressing and control plants, compared to WT plants (Fig. 6b). Amongst other four PR genes, PR-2 and PR-5 were strongly expressed only in LoMYB20s-expressing plants (Fig. 6b). PR-1, PR-2, and PR-5 are signature genes in plant defense responses to pathogenic infection [47], which also play important roles in various different abiotic stress responses [48,49,50,51]. The expression of PR genes is activated by the SA sensor protein NPR1, which stimulates transcriptional activity in response to pathogenic infection [52]. In addition, cold stimulation could also cause the induction of PR genes, which is mediated by a plasma membrane-tethered NACTF, NTL6, independently of NPR1-mediated SA signaling [53]. In the present work, the expression levels of NPR1 and NTL6 were unchanged in LoMYB20s-expressing plants (Additional file 1: Figure S2b), implying that PR-1, PR-2, and PR-5 may play roles in anther development that are distinct from the known biotic and abiotic stress responses.
Furthermore, we found that the expression levels of three cell-death-related protease genes, CEP2, SAG12, and MC9 were remarkably higher in LoMYB20s-expressing plants than in control plants (Additional file 1: Figure S2b, Fig. 6b). The CEP2 homolog CEP1 has been reported to be involved in the tapetal PCD process [24], and SAG12 was identified from leaves in the late stage of senescence that were visibly yellow [31]. Here, a gene upstream of SAG12, the senescence-associated TF gene WRKY53 [34], was also induced in LoMYB20s-expressing plants after DEX induction (Fig. 6b). Lastly, the cysteine-type peptidase gene MC9 is involved in the modulation of autophagy, to confine cell death to the appropriate target cells during Arabidopsis vascular xylem differentiation [33]. Taken together, the results suggested that LoMYB20s might have a function in the regulation of the PCD process.
Specific cells such as those comprising conducting vessels and fibers deposit a SW rich in cellulose, hemicellulose, and lignin, with lesser amounts of pectin [54]. In the anther endothecium, SW deposition is required for the generation of the tensile force necessary for stomium rupture to release pollen grains [11]. The indehiscence of lily anthers in LoMYB20s-silenced flowers (Fig. 4b), and the procumbent inflorescence stems of LoMYB20s-expressing Arabidopsis plants (Fig. 5b) suggested that either silencing or, conversely, ectopic expression of LoMYB20s might compromise SW deposition. Among many SWB regulator genes (Additional file 1: Figure S2c), the expression of MYB7, MYB32, and KNAT7 was dramatically induced in LoMYB20s-expressing plants (Fig. 6c). These three genes are direct targets of the secondary master switches of the SWB regulatory network, MYB46 and MYB83 [36,55]. But neither these two MYBs nor other genes directly downstream of them, such as MYB43, MYB7 and MYB32 are negative regulators in lignin biosynthesis [56], and KNAT7 and its interacting proteins OFP4 and BLH6 are also inhibitors of SWB [57,58]. Although there are another two genes, FRA8 and IRX15L, involved in glucuronoxylan biosynthesis were also induced by LoMYB (Fig. 6c), they are not the main enzymes in xylan biosynthesis [56]. So based on the symptom of the decreased inflorescence stem strength (Fig. 5b)

and the expression induction of the negative regulator genes in
LoMYB20s-expressing plants (Fig. 6c), we are inclined to believe that LoMYB20s negatively regulated SWB in anther development. Previous studies also discovered some negative regulators of SWB which were involved in the regulation of anther dehiscence [59,60,61], but how these negative regulators coordinate with positive regulators acting on anther wall development is yet to be identified and explained.
Plant hormones interact with each other through signaling crosstalk in multiple stress responses and developmental processes. A previous study reported that GA suppressed DELLAs to promoted the expression of DAD1 and LOX1, up-regulated JA production, and promoted the expression of MYB21, MYB24, and MYB57 [20]. In the present work, we found that LoMYB20s could promote the expression of both JA biosynthetic and GA-deactivating genes (Fig. 6d). This result implied an antagonistic relation between JA and GA in LoMYB20s mediated regulatory network. Previous studies have observed the antagonistic interaction of JA and GA in the regulation of plant growth and defense, and the synergistic interaction of JA and GA in regulation of stamen development [20,62]. The findings reported here suggested some possibility of this antagonistic JA-GA interaction in LoMYB20s regulated specific PCD-and SWB-related processes in anther development.
A biomechanical model describing anther opening incorporates the outer epidermal cell layer dehydration, and the endothecial layer uneven SWT in some model plant and crops [9]. In our previous work, we exhibited a reversible open and close process in mature anthers of lily controlled by the ambient humidity, and a LoPIP2-silencing induced anther indehiscent phenotype [28]. Here, we found the silencing of a LoMYB20s gene also induced lily anther indehiscent phenotype (Fig. 4), which might be related to natural PCD and SWB damage (Fig. 6). Accompanied with the development of genetic transformation system of lily [63,64], these genes could be good candidates for developing anther-indehiscent lilies by molecular breeding in the future.

Plant materials
Oriental hybrid lily cultivar 'Siberia' is cultured worldwide and is one of the most popular bouquets of corm varieties on the Chinese floral market [28]. The flowers of 'Siberia' were obtained from Beijing Shengsitong Eco-Technology Co., Ltd. (China). The flower stems were placed in water immediately after harvesting and delivered instantly to the laboratory. The flower stems were then re-cut underwater to a length of ~80 cm and placed in deionized water. Lily anther development was divided into four stages according to the distinctive colors visible at the anther surface: white, green, yellow, and purple [28].

Cloning of LoMYB20s genes
Total RNA was extracted from yellow anthers of lily using Trizol reagent (Invitrogen, USA). cDNAs for cloning the 5' and 3' ends of MYB were prepared from 1 μg of total RNA from lily anthers, using the SMART TM RACE cDNA Amplification Kit (Clontech, USA). A degenerate primer, 5'-KCWARRTGGGGRAAYAGGTGGTC-3', was used, together with "Universal Primer A Mix" provided in the kit, to amplify potential MYB 3' sequences in lily. Then the MYB gene-specific primer, 5'-GGACCCTCCAAACATTCTATATCTATCT-3', was designed as a reverse primer based on the obtained 3' sequence and used with "Universal Primer A Mix" to amplify the MYB 5' sequence. Finally, the complete sequence of the MYB gene was obtained using specific primers designed according to the 5' and 3' ends of MYB. Multiple sequence alignment was carried outusing the EBI ClustalW server (http://www.ebi.ac.uk/clustalw/) and visualized using BioEdit (Ibis Biosciences, Carlsbad, USA).
Phylogenetic and molecular evolutionary analysis was undertaken using MEGA version 5 [65].

Southern blot analysis of LoMYB20s
Southern blot analysis was carried out using the DIG Application Manual for Filter Hybridization (Roche, Switzerland). Genomic DNA was extracted from lily anthers using the CTAB method and digested with BamHI, EcoRI, HindIII, and XbaI. Ten μg of each digested product was separated on an agarose gel (0.8%) and then transferred onto a positively charged nylon membrane (Roche) by capillary transfer using 20×SSC buffer; the DNA was then UV-cross linked to the filter at a strength of

Reverse transcriptase polymerase chain reaction (RT-PCR) analysis
To determine the expression levels of the LoMYB20s gene in various different organs of lily plants, samples of leaf, stem, petal, ovary, stigma, anther, anther wall, and pollen were obtained from cut flowers (leaves and stems attached) that were about to open. In determining the expression pattern of the LoMYB20s gene in lily anthers at different developmental stages, the stages of anther development were distinguished by color [28]. To determine the expression levels of LoMYB20s and candidate related genes in transgenic Arabidopsis, the above-ground parts of Arabidopsis plants were  Table S1.

Virus-induced gene silencing (VIGS) of LoMYB20s in lily anthers
Silencing of LoMYB20s was performed using VIGS, with pTRV vectors (pTRV1 and pTRV2) [66]. A 426bp fragment was amplified from the 5' region of LoMYB20s using the primers 5'-gaggtaccTACCATGGACAAGAGAGTGAT-3' and 5'-ctggatcccgGCTCGCTTGGCTCGTG -3'. The fragments were inserted into the KpnI and BamHI restriction sites of pTRV2 to form the pTRV2-LoMYB20s construct. pTRV2-LoMYB20s, pTRV1, and pTRV2 were transformed individually into Agrobacterium tumefaciens GV3101. For infiltration, Agrobacterium suspensions containing a 1:1 (v/v) ratio of either (i) pTRV1 and pTRV2-LoMYB20s or (ii) pTRV1 and pTRV2 (control) were prepared. Lily plants were infected when the flower buds were ~4 cm in length and contained white anthers at an early stage of development [28]. Approximately ~15 days after infiltration, the flowers were photographed, and the anthers were collected for characterization.

Arabidopsis transformation and morphological observations
To transform the LoMYB20s gene into Arabidopsis, we first amplified the LoMYB20s ORF, using as primers: forward, 5'-aaactcgagACTCGAGATGGACAAGAGAGTGATCCCT-3'; reverse, 5'-aaaactagtCGTCTCCATTGAAAGACTGCATAG-3'. The resultant product was ligated into the pTA7001G vector using the XhoI and the SpeI sites. The constructed vectors were then introduced into Agrobacterium tumefaciens strain GV3101. Transformations with Arabidopsis were performed using the flower-dip method, as described by Clough and Bent [67].

Additional File Information
Additional file 1: Figure S1. Growth phenotypes of transgenic Arabidopsis lines of DEX::LoMYB20s plants. Figure S2. The semi-quantitative PCR detection of related genes. Table S1. Primer sequences used in this study.