The Suppression of a R2R3-type LoMYB gene damaged the anther dehiscence in lily CURRENT STATUS: UNDER REVISION

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 LoMYB from oriental lily ( Lilium sp. ‘Siberia’). LoMYB mainly expressed in anther wall during the late stages of lily anther development. Suppression of LoMYB by virus-induced gene silencing (VIGS) in lily led to a failure of the anthers to dehisce. Induction of LoMYB in DEX:: LoMYB transgenic Arabidopsis by spraying dexamethasone (DEX), caused the rosette leaves turning yellow and the inflorescences becoming procumbent and infertile. And the induced genes in Arabidopsis include developmental programmed cell death (PCD) and secondary wall biosynthesis (SWB) related genes. These results suggested that MYB regulated anther dehisce possibly through promoting the PCD and SWB processes in cells. Conclusions Our results demonstrated the indispensable role of LoMYB in lily anther dehisce, discovered LoMYB participated in PCD and SWB regulatory processes, and provide a possibility of using LoMYB silencing to produce anther-indehicent lilies.


Abstract
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 LoMYB from Conclusions Our results demonstrated the indispensable role of LoMYB in lily anther dehisce, discovered LoMYB participated in PCD and SWB regulatory processes, and provide a possibility of using LoMYB silencing to produce anther-indehicent lilies.

Background
Anther development is a multistage process from cellular differentiation to localized degeneration [1]. During the early stage of development, anther cell and tissue differentiation occurs, so that by the end of this phase, the anther contains most of its specialized cells and tissues, comprising the outer epidermis, endothecium, middle cell layer, and the tapetum surrounding the innersporogenous cells [2,3]. Subsequently, during the middle stage of development, the anther enlarges, and this is accompanied by tapetal development, pollen mother cell meiosis, and septum and stomium formation; then, during the late stage of development, tapetal degeneration is coordinated with microspore maturation and pollen wall formation, endothecium expansion and subsequent deposition of ligno-cellulosic thickening, and with degeneration of the septum and stomium [3,4]. Finally, pollen swelling and endothecium dehydration induces anther dehiscence and pollen release as the flower opens [5,6].
During the late stage of anther development, tapetal degeneration plays a crucial role in pollen maturation by contributing nutrients, enzymes, and sporopollen in precursors for pollen wall synthesis and pollen coat deposition [7]; and septum and stomium degeneration and endothecium secondary wall (SW) thickening play important roles in anther dehiscence and pollen release, providing the mechanical force for the anther wall to roll outward and facilitating the opening of the anther [6]. 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 [1,8].
R2R3-MYBs comprise one of the largest families of plant TFs and are important regulators playing diverse roles in anther development [9,10]. In Arabidopsis, MYB21, MYB24, and MYB57 are critical components in the JA-mediated transcriptional cascade for stamen development and regulate over all filament elongation and anther dehiscence [11,12]. In addition, the anther-specific gene MYB26 governs SW thickening in the endothecium and functions upstream of the central TF genes involved in SW formation, including NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 ( NST1) and NST2 [13,14]; the GAMYB-like genes MYB33/MYB65 facilitate tapetal and pollen development [15]; and MYB80 is required for tapetal cell death (TCD), callose dissolution, exine formation, and pollen development in Arabidopsis [16,17] and are functionally conserved in crops such as cotton, lily, and Brassica [18,19,20].
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 LoMYB from lily, which is specifically expressed in the late phase of anther development.
Investigation of the effect of LoMYB on lily anthers showed an effect on anther dehiscence, and it was concluded that this might result from a cessation of PCD-related anther tissue degeneration and an inhibition of SW development, possibly via a JA/GA (jasmonate/gibberellin) co-mediated regulatory pathway.

Results
LoMYB 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 [11,12]. 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 LoMYB and the accession number KT759161. The full-length nucleotide sequence of LoMYB was 807 bp, encoding a deduced protein sequence of 190 amino acids.
LoMYB has two MYB DNA-binding domains (Fig. 1a), and is a R2R3-type MYB protein [10]. A BLAST homology search of the sequence of LoMYB 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 [10]. LoMYB 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 LoMYB in the lily genome Southern hybridization was used to identify whether there were multiple copies or close homologs of LoMYB in the lily genome. The full-length sequence of LoMYB 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 LoMYB sequence, the results suggested that LoMYB is a single-copy gene, with no close homologs in the lily genome.
LoMYB is mainly expressed in the late stages of lily anther development Levels of expression of LoMYB were examined in a range of tissues of the lily plant, including stem, leaf, petal, ovary, stigma, filament, anther, anther wall, and pollen. LoMYB 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 [21]. A low level of expression of LoMYB was detected at the white and green stages, which then increased at the yellow and purple stages, and then declined following anther dehiscence (Fig. 3b). These results suggested that LoMYB probably functioned during the late phase of anther development.

LoMYB silencing inhibits anther development and dehiscence
To examine the possible role of LoMYB in anther dehiscence, we silenced LoMYB in lily using virus-induced gene silencing (VIGS) [21]. Total RNA was extracted from anthers of lily plants infected with Agrobacterium tumefaciens carrying pTRV-LoMYB or the pTRV empty vector and analyzed by semi-quantitative RT-PCR (Fig. 4a). LoMYB was successfully silenced in the anthers of six lines of lilies (6/100), and representative LoMYB-silenced flowers of lily are shown in Fig. 4b. We observed that, in the LoMYB-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  Figure S1). Under DEX treatment, the control plants grew, flowered, and bore seed normally, whereas the LoMYB-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 LoMYB caused early senescence of the transgenic plants, and severely influenced inflorescence development in Arabidopsis.

Arabidopsis
In an attempt to explain the phenotypes of LoMYB-silenced lily anthers and LoMYBexpressing 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 LoMYB-expressing Arabidopsis, the expression of LoMYB and its closest MYB homologues was initially examined (Fig. 6a, Additional file 1: Figure S2a). As anticipated, the expression of LoMYB 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 LoMYB expression. Then, the expression of the closest homologues of LoMYB 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 two MYBs [11] 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 LoMYB 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 DEXinduced plants. Their expression was then precisely quantitated using qRT-PCR, which revealed dramatically higher levels of expression, relative to controls, in the LoMYBexpressing plants (Fig. 6b). These genes included, amongst others, three pathogenelicited PCD-associated genes, PR-1, PR-2, and PR-5 [22], a KDEL-tailed serine protease (CysP) gene, CEP2 [23], a senescence-associated CysP gene, SAG12 [24], a vascular xylem autophagy modulator gene, MC9 [25,26], and a senescence-associated TF gene, WRKY53 [27].

Genes of JA and GA metabolism were altered in transgenic Arabidopsis
Among genes downstream of LoMYB 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 JMT34], and three genes of GA deactivation, GA2ox4, GA2ox6, and GA2ox8 [35], were induced in LoMYB-expressing plants compared to the control plants after DEX treatment (Fig. 6d). These results suggest that LoMYB could be involved in JA/GAassociated stamen developmental regulation.

Discussion
Regarding its homology to MYB proteins in Arabidopsis, LoMYB is most closely related to MYB57, MYB21, and MYB24 (Fig. 1b). MYB57, MYB21, and MYB24 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 [12]. In contrast to the shorter filaments and petals displayed in the delayed dehiscence phenotypes exhibited by myb21, myb21myb24, or myb21myb24myb57 mutants [11,36], only slender and indehiscent anthers were observed in LoMYB-silenced lily flowers (Fig. 4b). In transgenic LoMYBexpressing Arabidopsis plants, MYB21 expression was only slightly influenced, compared to MYB24 and MYB57, which were strongly induced (Additional file 1: Figure S2, Fig. 6a). Among these genes, only PAL2 and TPS21 were significantly induced in LoMYB-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 [36], but none of them showed significant change in LoMYB-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, LoMYB regulated anther dehiscence more specifically than its homologs.
Given that LoMYB-expressing Arabidopsis plants exhibit a phenotype of early senescence after DEX induction (Fig. 5a), it is reasonable to suspect that LoMYB 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 MYB80 [16,17], MYB33, MYB65 [15], MYB101 [38], and MYB30 [39]. None of these genes showed changes in expression in LoMYB-expressing plants after DEX induction (Additional file 1: Figure   S2b). But one of the downstream targets of MYB30, PR-1 [40] showed strongly altered expression in both LoMYB-expressing and control plants, compared to WT plants, with the expression being a little higher in LoMYB-expressing plants than in control plants (Fig. 6b).
Amongst other four PR genes, PR-2 and PR-5 were strongly expressed only in LoMYBexpressing plants (Fig. 6b). PR-1, PR-2, and PR-5 are signature genes in plant defense responses to pathogenic infection [41], which also play important roles in various different abiotic stress responses [42,43,44,45]. The expression of PR genes is activated by the SA sensor protein NPR1, which stimulates transcriptional activity in response to pathogenic infection [46]. In addition, cold stimulation could also cause the induction of PR genes, which is mediated by a plasma membrane-tethered NACTF, NTL6, independently of NPR1mediated SA signaling [47]. In the present work, the expression levels of NPR1 and NTL6 were unchanged in LoMYB-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 LoMYB-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 [17], and SAG12 was identified from leaves in the late stage of senescence that were visibly yellow [24]. Here, a gene upstream of SAG12, the senescence-associated TF gene WRKY53 [27], was also induced in LoMYB-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 [26]. Taken together, the results suggested that LoMYB may have a function in the regulation of the PCD process.
Specific cells such as those comprising conducting vessels and fibers deposit a secondary wall (SW) rich in cellulose, hemicellulose, and lignin, with lesser amounts of pectin [48]. In the anther endothecium, SW deposition is required for the generation of the tensile force necessary for stomium rupture to release pollen grains [14]. The indehiscence of lily anthers in LoMYB-silenced flowers (Fig. 4b), and the procumbent inflorescence stems of LoMYB-expressing Arabidopsis plants (Fig. 5b) suggested that either silencing or, conversely, ectopic expression of LoMYB 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 LoMYB-expressing plants (Fig. 6c) [50,51]. OFP4 was dramatically induced, and BLH6 slightly induced in LoMYB-expressing plants (Fig. 6c). The procumbent inflorescence stems and the induction of KNAT7, BLH6, and OFP4 expression in LoMYB-expressing plants (Fig. 6c) suggested that LoMYB may regulate SWB through promoting the expression of the genes involved in the TALE-OFP protein complex.
Our results also indicated that SW formation involved the effector genes IRX7, IRX15L, and QRT2 [31,32,33,52], all of which were sharply induced in LoMYB-expressing plants compared to control plants (Fig. 6c). These results provided further evidence that LoMYB participates in SWB regulation, and suggested a connection between the SWB regulators MYB7, MYB32, the KANT7/BLH6 /OFP4 complex and the SWB effectors, IRX7, IRX15L, and QRT2.
Just as SW deposition is directly associated with PCD during the differentiation of xylem vessels (trachearyelements, TEs) [53], during anther development, pollen grain maturation, and pollen wall formation are directly associated with tapetal degeneration, and endothecium SW thickening is associated with the immediate PCD of the endothecium and septum. It is possible that LoMYB act like VASCULAR-RELATED NAC-DOMAIN 6 ( VND6) and VND7, which not only have a function in SW formation, but also are involved in PCD in TEs [54,55].
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 [11]. In the present work, we found that LoMYB 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 LoMYB 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 [11,56]. The findings reported here suggested some possibility of this antagonistic JA-GA interaction in LoMYB 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 secondary thickening in some model plant and crops [57,58]. In our previous work, we exhibited a reversible open and close process in mature anthers of lily controlled by the ambient humidity, and a LoPIP2silencing induced anther indehiscent phenotype [21]. Here, we found the silencing of a LoMYB gene also induced lily anther indehiscent phenotype (Fig. 4), which might be related to cell death and second wall thickening damage (Fig. 6). Accompanied with the development of genetic transformation system of lily [59,60], these genes could be applied to develop anther-indehiscent lilies by molecular breeding in the future.

Plant materials
Oriental hybrid lily cultivar 'Siberia' is cultured worldwide and is currently one of the most important bouquets of corm varieties on the Chinese floral market [21]. 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 [21].

Cloning of LoMYB 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 genespecific 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 [61].

Southern blot analysis of LoMYB
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 Arabidopsis were performed using the flower-dip method, as described by Clough and Bent [63]. analysis. qRT-PCR was performed using 2×Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific, USA) and the Mx3005P QPCR System (Agilent, USA), and relative mRNA levels were calculated using the 2 -ΔΔCT method [64]. Three biological replicates were performed for qRT-PCR. The ACTIN7 gene was used as a reference for normalization. All primers used for RT-PCR and qRT-PCR are listed in Additional file 1: Table S1.