Expression of MYB transcription factor gene ZmMYB59 affects seed germination in Nicotiana tabacum and Oryza sativa

Background: MYB transcription factors are involved in many biological processes, including metabolism, development and responses to biotic and abiotic stresses. In our previous work, a new MYB transcription factor gene, ZmMYB59 was induced by deep sowing and down-regulated during maize seed germination via Real-Time PCR. However, there are few reports on seed germination regulated by MYB proteins and the functions of ZmMYB59 remain unknown. Results: In this study, to examine its functions, Agrobacterium-mediated transformation was exploited to generate ZmMYB59 transgenic tobacco and rice. In T 2 generation transgenic tobacco, germination rate, germination index, vigor index and hypocotyl length were signicantly decreased by 25.0~50.9%, 34.5~54.4%, 57.5~88.3% and 21.9~31.3% compared to wild-type (WT) lines. In T 2 generation transgenic rice, germination rate, germination index, vigor index and mesocotyl length were notably reduced by 39.1~53.8%, 51.4~71.4%, 52.5~74.0% and 28.3~41.5%, respectively. On this basis, relative physiological indicators were determined. The activities of catalase, peroxidase, superoxide dismutase, ascorbate peroxidase and proline content of transgenic lines were signicantly lower than those of WT, suggesting that ZmMYB59 reduced their antioxidant capacity. As well, ZmMYB59 expression extremely inhibited the synthesis of gibberellin A1 (GA 1 ) and cytokinin (CTK), and promoted the synthesis of abscisic acid (ABA) concurrently, which implied that seed germination was repressed by ZmMYB59 in hormone levels. Furthermore, cell length and cell number of hypocotyl/mesocotyl in transgenic tobacco and rice were notably decreased. Conclusions: Taken together, it proposed that ZmMYB59 plays a negative regulatory role during seed germination in tobacco and rice, which also contributes to illuminate the molecular mechanisms regulated by MYB transcription factors.


Background
Seed germination is a remarkably pivotal stage in the biological clock of plants [1], involving the fast absorbing water by the dry seeds until all of the seed organs are hydrated thoroughly; a dynamic equilibrium in which the water potential of the seed remains unchanged; and a phase of rapid combination water for radical elongation [2][3][4]. For most plants, the germination stages are the most delicate to biotic and abiotic stresses. On that account, the investigation into seed germination is recommended to assess and breed the ecological adaptation of the crops. Recently, some researches have documented that MYB transcription factors have noteworthy in uences on this developing phases [5][6][7][8].
MYB, the largest transcription factor family in plants, is widely distributed in both monocotyledons and dicotyledons [9,10]. MYB proteins are characterized by a highly conserved DNA-binding domain: the MYB domain about 52 amino acids, the C-terminal region alters strikingly from one MYB protein to another, thereby allowing MYB superfamily to perform a considerable assortments of structures and functions [6,11]. Depending on the number of conserved motifs, the superfamily is divided into four classes: R1-MYB, R2R3-MYB, R1R2R3-MYB and 4R-MYB [12]. The functions of MYB proteins have been probed in plentiful plant species such as Arabidopsis, maize, rice, petunia, snapdragon, grapevine, poplar and apple [9], involving the regulation of cell differentiation, plant development, organ morphogenesis, phytohormone response, stress tolerance, secondary metabolism [13,14].
In view of the regulation of seed germination by MYB transcription factors, AtMyb7 mutants produced a lessened lateral root length, playing a key role in establishment of homeostasis during seed germination in Arabidopsis [6]. RSM1, an Arabidopsis MYB protein could modulate seed germination in response to ABA and salinity [7]. LcMYB2 involved the potential to increase root growth to enhance drought tolerance during sheepgrass seed germination [8]. However, there are relatively few reports on seed germination regulated by MYB proteins and the explicit mechanisms remain unidenti ed.
In our previous study, a new MYB transcription factor gene named ZmMYB59, was cloned from the B73 inbred line. Affymetrix GeneChip was employed to analyze the gene expression patterns of mesocotyl tissue during maize seed germination at 20 cm sowing depth, indicating that MYB genes were signi cantly expressed [15]. Thereafter, Real-Time PCR showed the expression of ZmMYB59 in maize mesocotyl was fairly down-regulated during seed germination [16]. In this study, ZmMYB59 transgenic tobacco and rice were produced by genetic transformation. Afterwards, germination experiment, antioxidant capacity, cellular morphology and phytohormone content were measured. The objective of this study was to further investigate the functions of ZmMYB59 during seed germination in exogenous expressed tobacco and rice, which will also contribute to elucidate the regulatory mechanisms by MYB transcription factors affecting seed germination.

Results
Veri cation of the integration of the ZmMYB59 gene into the tobacco and rice genomes The expression vector pCAMBIA3301-Bar-ZmMYB59 construct was transferred into immature embryos to gain transgenic tobacco and rice. The regeneration of somatic embryos and their conversion into plants was attempted to each transgenic line. The different stages of plant regeneration of stable transgenic lines were described in Fig 1. T 0 generation transgenic plants were continually self-pollinated until T 2 generation. Then, to validate the presence of the ZmMYB59 gene, T 2 generation transgenic tobacco and rice were tested by PCR ampli cation, wild-type was as negative control, ZmMYB59 gene was as positive control. PCR products were tested by agarose gel electrophoresis. In tobacco, the result of electrophoresis showed that 18 of 22 transgenic lines were consistent with the positive control (Additional le 1: Figure  S1). In rice, the results suggested that 16 of 19 lines were successfully transformed (Additional le 1: Figure S2). During all electrophoretic bands, three over-expression lines were selected to be employed in this study (OE1, OE2, OE3 in tobacco, OE2, OE4, OE6 in rice), which was showed in Fig 2a, 2b.

Effect of ZmMYB59 expression on seed germination
The results showed that ZmMYB59 signi cantly improved seed germination among three independent homozygous transgenic lines. This improvement was re ected on higher germination rate, germination index, vigor index, as well as longer hypocotyl/mesocotyl length. In T 2 generation transgenic tobacco, germination rate, germination index, vigor index and hypocotyl length were decreased by 25.0~50.9%, 34.5~54.4%, 57.5~88.3% and 21.9~31.3% compared to WT lines ( Table 2, Fig 3a). In T 2 generation transgenic rice, the corresponding indexes were reduced by 39.1~53.8%, 51.4~71.4%, 52.5~74.0% and 28.3~41.5%, respectively ( Table 2, Fig 3b). The above results suggested that ZmMYB59 played a negative regulatory role in the process of seed germination in both transgenic tobacco and rice.

Effect of ZmMYB59 expression on antioxidant capacity
To investigate whether ZmMYB59 expression decreases antioxidant capacity, the contents of malondialdehyde (MDA), proline content and the activities of CAT, POD, SOD and APX were measured (Table 3). In T 2 generation transgenic tobacco, MDA content was enhanced by 5.7~21.5% and proline content was reduced by 32.9~60.6% compared to WT lines. In T 2 generation transgenic rice, MDA content was increased by 4.3~8.0% and proline content was decreased by 6.6~10.9%. Moreover, the activities of CAT, POD, SOD and APX of transgenic tobacco were signi cantly decreased by 32.3~46.2%, 18.0~25.3%, 9.8~18.9%, 19.8~29.0%, respectively. In transgenic rice, the above enzymatic activities were decreased by 8.3~12.8%, 9.0~19.4%, 24.8~43.5%, 36.5~59.6%, respectively. It could be safely documented that ZmMYB59 could decrease antioxidant capacity of transgenic tobacco and rice, which was generally consistent with the results of germination experiment.

Effect of ZmMYB59 expression on cellular morphology
Considering that ZmMYB59 reduced hypocotyl/mesocotyl length, cellular morphology of hypocotyl/mesocotyl in tobacco and rice was observed in this experiment to determine whether and how MYB affects cell proliferation and elongation. This is indeed the case (Fig 4, Table 4). In T 2 generation transgenic tobacco, cell number and cell length of hypocotyl were signi cantly decreased by 12.8~22.2% and 21.7~42.7% compared to WT lines (Fig 4a, 4b). In T 2 generation transgenic rice, cell number and cell length of mesocotyl were signi cantly reduced by 20.0~28.2% and 10.8~17.6% (Fig 4c, 4d). The results suggested the decrease of hypocotyl/mesocotyl length caused by ZmMYB59 was attributed to the inhibition of cell growth including cell number and cell length.

Effect of ZmMYB59 expression on content of phytohormone
To investigate the in uence of ZmMYB59 expression on endogenous phytohormones during seed germination, the contents of ABA, IAA, GA 1 , GA 3 , GA 4 , CTK in hypocotyl/mesocotyl were determined. In T 2 generation transgenic tobacco, compared to WT lines, the contents of endogenous GA 1 , GA 3 , GA 4 , IAA, CTK were reduced by 21.1~39.2%, 18.7~29.9%, 3.3~15.4%, 3.4~7.5% and 27.9~44.8%, whereas the content of ABA was increased by 23.8~43.9% (Table 5). In T 2 generation transgenic rice, the contents of endogenous GA 1 , GA 3 , GA 4 , IAA and CTK were decreased by 29.4~47.6%, 14.9~22.3%, 15.4~24.3%, 5.7~10.0% and 15.7~37.8%, whereas the content of ABA was increased by 17.9~26.9% (Table 5). Among them, the changes of endogenous GA 1 , CTK and ABA reached signi cant level while there were no signi cant changes in those of endogenous GA 3 , GA 4 and IAA. These results indicated that the inhibiting effect of ZmMYB59 might be ascribed to the promotion of endogenous GA 1 and CTK synthesis and the inhibition of endogenous ABA synthesis.

Discussion
Seed germination is directly related to eld establishment and crop yield. There are some documents that some transcription factors normally de ned as proteins are capable of mobilizing or suppressing plant growth [17]. Nonetheless, the contributions of MYB transcription factors during seed germination have not yet been functionally characterized. In our previous study, to elucidate the molecular mechanisms of maize seed germination at 20 cm sowing depth, gene expression of mesocotyl was analyzed by Affymetrix GeneChip [15]. The results demonstrated that there was the highest expression level of MYB genes, accounting for 8.15% of total number of transcription factor family [15].
However, its functions of MYB protein on seed germination have not been investigated thoroughly. The was found to be involved in plant growth and stress responses by acting as a negative regulator of Ca 2+ signaling and homeostasis [10]. These reports suggested that MYB59 transcription factor might play important roles in plant growth, however, its functions on seed germination remained unclear.
The sequence of ZmMYB59 gene was rst issued in 2009. Through BLASTp of NCBI, comparing the amino acid sequence of ZmMYB59 protein [GenBank: ACG37097.1] with AtMYB59 protein [GenBank: NP_200786.1], sequence identity between them attained 53.65%, suggesting that ZmMYB59 is indeed the homologous gene of AtMYB59. We speculated that ZmMYB59 might also play negative regulation and made some research to prove our conjectures. The expression of ZmMYB59 in maize mesocotyl was remarkably down-regulated after incubation for 6~8 d [16]. It was well known that seed germination was attributable primarily to the elongation of the mesocotyl and the rst internode [22,23]. In like manner, AtMYB30 was highly expressed in brassinosteroid pathway to manipulate hypocotyl cell prolongation during Arabidopsis thaliana seed germination [24]. Consequently, mesocotyl/hypocotyl length was an essential index to in ect germination. In this study, germination experiments revealed that hypocotyl/mesocotyl length of transgenic tobacco and rice was signi cantly lower than wild-type lines. To further detect the effect of ZmMYB59 expression on cell growth, cell morphology of hypocotyl/mesocotyl was observed. Our results suggested that the reduction of hypocotyl/mesocotyl length caused by ZmMYB59 was due to reducing both cell length and cell number among three transgenic lines. Meanwhile, proline, under extreme adversity, will protect plant protein from osmotic stress [31]. In this study, compared to the wild-type lines, MDA content was increased and proline content and the activities of CAT, POD, SOD, APX were decreased in ZmMYB59 transgenic tobacco and rice. These results implied that ZmMYB59 expression could inhibit seed germination by reducing antioxidant capacity.
The phytohormones GA, CTK, ABA were reported to play antagonistic roles in the control of seed germination [30]. GA released dormancy and stimulates seed germination by enhancing the proteasomemediated destruction of RGL2 (RGA-LIKE2), a key DELLA factor repressing germination [32,33]. ABA biosynthesis was associated with the maintenance of seed dormancy, leaf senescence and inhibited germination [34,35]. Cytokinin (CTK) regulated diverse processes from embryonic development to adult plant growth [36]. It can be safely concluded that some MYB transcription factors played important roles in phytohormone regulation. For example, AtMYB60 and AtMYB96 could synergistically control stomatal aperture, drought and disease resistance by ABA signal pathway [9]. GAMYB expression in the rst internode was substantially increased by GA 3 application in a wheat variety, named Hong Mang Mai [37].
AtMYB7 negatively regulated ABA-induced inhibition of seed germination by blocking the expression of a bZIP transcription factor ABI5 [6]. Overexpression of OsMYBR1 conferred improved drought tolerance and decreased ABA sensitivity in rice [38]. CLAU was a MYB transcription factor that modulated leaf morphogenesis by constraining the morphogenetic potential, in part due to attenuation of CTK signaling [39].
Gibberellin (GA) was essential intermediate in the stimulation of seed germination, including GA 1 , GA 3 , GA 4 [40,41] . In our previous study, discovering that the expression level of ZmMYB59 in maize mesocotyl was inhibited when seed soaked with 10 -5 M GA 3 , and in plumules and roots were strongly increased by ABA [13]. Furthermore, after GA 3 soaking, endogenous GA 3 , GA 4 and ABA were controlled at a relatively low level, but endogenous GA 1 was controlled at a relatively high level in seed embryos of Masson pine [41]. The results showed that exogenous GA 3 improves seed germination through lowering the ABA level and stimulating GA 1 biosynthesis, which indicated that endogenous GA 1 might play more important roles during seed germination rather than endogenous GA 3 , GA 4 [41]. In this study, our results showed that ZmMYB59 expression decreased the total levels of endogenous GA 1 , GA 3 , GA 4, IAA, CTK and increased the level of endogenous ABA, but only endogenous GA 1 , CTK and ABA had signi cant changes.
Therefore, it could be concluded that the inhibitory effect of ZmMYB59 was attributed to endogenous GA 1, but not GA 3 and GA 4 . Taken together, the results suggested that endogenous GA 1 could play more important roles during seed germination in ZmMYB59 transgenic tobacco and rice, which was generally consistent with the research results of embryos of Masson pine according to the study of Zhao et al. (2014) [41] In summary, the molecular mechanisms regulated by ZmMYB59 gene during seed germination of tobacco and rice can be elucidated in Fig 5. Expressing ZmMYB59 inhibited the synthesis of endogenous GA 1 and CTK, and promoted the synthesis of endogenous ABA. This effect reduced the antioxidant capacity, which directly affected cell growth. All above effects together inhibited hypocotyl/mesocotyl elongation, which suggested that ZmMYB59 gene was a negative regulatory factor during seed germination in tobacco and rice. In our future work, genetic transformation of ZmMYB59 gene in maize will be performed to further validate its functions. Gene knockout is advised as an effective strategy to breeding new maize varieties, which improve seed germination.

Conclusions
The results reported here demonstrated that ZmMYB59 heterogenous expression in tobacco and rice had a negative effect on seed germination by inhibiting the synthesis of GA, CTK and IAA and promoting the synthesis of ABA, then reducing the contents of proline and the activities of CAT, POD, SOD, APX, as a result, repressing cell proliferation and elongation. The result also con rms that endogenous GA 1 may play more important roles during seed germination rather than GA 3 , GA 4 . Our ndings suggest that ZmMYB59 plays a negatively regulatory role in tobacco and rice, which will contribute to elucidate the mechanisms of seed germination regulated by MYB transcription factors and also provides a key gene affecting seed germination. Considering the fact that sowing covered with soil was generally required in crop production, seeds of tobacco and rice were sowed at the depth of 1 cm and 4 cm, respectively. After incubation for 2 d, the number of germinated seeds was recorded each day until 14 d. Germination rate, germination index, vigor index were determined according to following formulas:

Methods
Germination rate = n 1 within 14 days / n 2 × 100%. Here n 1 is the number of germinated seeds; n 2 is the number of tested seeds.
Vigor index = ∑ (Gt / Dt) × S. Here Gt is corresponding number of seeds germinated in the t day; Dt is time corresponding to Gt in days; S is the average length of 10 seedlings.

Determination of antioxidant capacity
Malondialdehyde (MDA) content was measured by using 2-thiobarbituric acid [27]. Fresh leaves 0.5 g were ground with 5 ml 0.6% TBA in 10% trichloroacetic acid. The mixture was heated at 100 °C for 15 min and then cooled in ice bath. Finally, read the absorbance at 450, 532 and 600 nm. computational formula was as follows: µmol MDA g −1 fresh weight (FW) = 6.45 (OD 532 − OD 600 ) − 0.56 OD 450 . The proline content was assayed from freeze dried leave material, using a 3% sulfosalicylic and ninhydrin extraction buffers [42]. Dry leaves 0.04 g was homogenized with 3% sulfosalicylic acid and centrifuged at 3000 g/min for 10 min. 200 µl supernatant was mixed with 400 µl reagent mixture and heated in sealed test tubes at 100 °C for 1 h. After cooling down, 4 ml toluene was added to each sample. Proline content was measured by spectrophotometer at 520 nm.
Until the seedlings grew out of 4~5 cm, the activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX) were measured by employing 0.5 g samples in 5 mL extraction buffer containing 0.05 M phosphate buffer [30,43]. CAT was determined spectrophotometrically based on the decrease in absorbance of H 2 O 2 at 240 nm. POD was measured as the absorbance at 470 nm. SOD was assayed by measuring the ability of the enzyme extract to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). APX was assayed from the decrease in absorbance at 290 nm as ascorbate was oxidized [44].

Observation of cell morphology
Hypocotyl/mesocotyl of wild-type and transgenic seedlings was cut longitudinally and the cell sections were made to determine the changes of cell length and cell number. Firstly, the middle position of hypocotyl/mesocotyl was cut and xed with 25% glutaraldehyde stationary solution initially. Next ve visual elds were randomly selected and cell length of hypocotyl/mesocotyl was measured by calibrated eyepiece, and cell number was counted by pictures.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
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Availability of data and materials
All the necessary data generated or analyzed during this study have been included in this article.

Competing interests
All the authors declared that they had no con ict of interest. Tables Table 1 The nucleotide sequences of the primer pairs used to identify the PCR products of expressed ZmMYB59 gene primer sequence 5 -3 T m (℃) Product size (bp) ZmMYB59-F ATTGAGCTCCATGCTCGGTG 60 580 ZmMYB59-R TAGCTGAGTGGCCTGACCAA 60   Means with standard deviations that do not followed by the same lower case letter between OE and WT lines significantly differ by ANOVA analysis at 5% level of significance. Note: WT and OE represent wild-type and ZmMYB59 transgenic plants respectively. Means with standard deviations that do not followed by the same lower case letter between OE and WT lines significantly differ by ANOVA analysis at 5% level of significance.