Abstract
Grape (Vitis vinifera) is among the world’s most important fruit crops and is a commonly used woody plant for genomics and post-genomics research. NAC transcription factors play central roles in plant growth and development, floral organ morphogenesis, and responses to biological stress. It is therefore important to identify key transcription factors from grape and clarify their mechanisms of action to generate genetic resources for grape molecular improvement. Our research group previously cloned a NAC transcription factor from V. vinifera ‘Yatomi Rosa’ [drought and leaf roll gene 1 (DRL1)] and demonstrated that it caused dwarfing of tobacco (Nicotiana benthamiana) plants when overexpressed. In the present work, we demonstrate that overexpression of DRL1 in transgenic tobacco delays flowering time and markedly reduces pollen viability. Furthermore, crosses between male DRL1 transgenic tobacco and female wild-type tobacco exhibit substantially lower fruit set, fruit and seed weights, fruit and seed shape indices, and seed germination rates than selfed wild-type plants or crosses with a transgenic female parent. DLR1 overexpression strongly influences flowering time and reproduction in transgenic tobacco, primarily through its effects on pollen development. These results provide a foundation for further functional characterization of DLR1 in grape.
Grape (Vitis vinifera) is a deciduous vine of the Vitaceae family; it is one of the most important fruit crops in the world, with a large cultivation area and a wide variety of uses. It was the first fruit crop with a fully sequenced genome (Jaillon et al., 2007), which led to improvements in fruiting ability and quality; development of new cultivars through genetic engineering; and acceleration of resource evaluation and breeding (Yu et al., 2020).
NACs are a plant-specific transcription factor family whose name is derived from the first letters of the petunia (Petunia hybrida) NAM gene and the Arabidopsis thaliana ATAF1/2 and CUC2 genes (Riechmann et al., 2000). The NAC family is one of the largest gene families in plants (Guo et al., 2015; Hu et al., 2010; Olsen et al., 2005), and its members are involved in various stages of plant growth and development, including the formation of apical meristems (Souer et al., 1996), embryonic development (Kunieda et al., 2008), floral organ morphogenesis (Sablowski and Meyerowitz, 1998; Sanders et al., 1999), flowering time regulation (Guo et al., 2018), lateral root initiation (Sohrabi et al., 2017), and leaf senescence (Zhu et al., 2019).
Research on the NAC family in grape has focused mainly on the functional characterization of specific genes (Fernandez et al., 2006; Tello et al., 2015). For example, VpNAC1 from the wild Chinese grape Vitis pseudoreticulata was shown to respond to external pathogens and exogenous hormonal signals, and its overexpression in tobacco (Nicotiana benthamiana) enhanced resistance to powdery mildew (Erysiphe cichoracearum) and Phytophthora parasitica var. nicotianae (Zhu et al., 2012). Seventy-four NAC transcription factors were identified in grape based on a genome-wide analysis, and the functions of a small number of them were demonstrated, mainly regarding stress response and morphological development (Wang et al., 2013). Le Hénanff et al. (2013) found that VvNAC1 was highly expressed in fruit pulp, leaves, and flowers during the late stage of development, suggesting that its function may be related to plant senescence and/or flowering. NAC26 from Vitis amurensis promoted tolerance to drought stress by regulating jasmonic acid in A. thaliana (Fang et al., 2016). Various VvNAC26 polymorphisms and their combination influenced berry size variation in grape (Tello et al., 2015). In our previous work, the NAC transcription factor gene DRL1 (GenBank no. XP-002281816) was identified from V. vinifera ‘Yatomi Rosa’ and shown to be associated with dwarfing and rolled leaf phenotypes (Yan et al., 2016). DRL1 also appears to have a role in senescence, because its overexpression in tobacco plants significantly delayed leaf senescence (Zhu et al., 2019). Here, we expanded upon this preliminary work by studying the effects of DRL1 on tobacco flowering and fruiting. Specifically, we investigated flowering time, observed the phenotypes of hybrid fruit, and assessed the pollen viability of DRL1 transgenic tobacco. We also transformed tobacco with a DRL1pro::GUS fusion construct to visualize DRL1 overexpression in tobacco floral tissues. The results shed light on the functions of DRL1 and add to our theoretical understanding of NAC transcription factor regulation.
Materials and Methods
Production of transgenic tobacco.
Grapevine is known to be one of the most refractory plants for transformation (Dutt et al., 2007; Duval et al., 2002; Maqsood et al., 2015). Here, we performed heterologous overexpression of DRL1 in transgenic tobacco because of its ease of transformation and regeneration and its short growth period relative to grape. Yatomi Rosa (a very early-maturing red table grape cultivar) was grown in the grape germplasm resource orchard of the Henan Institute of Science and Technology, Xinxiang, Henan, China. The full-length DRL1 gene was cloned from leaves of ‘Yatomi Rosa’ and inserted into the fusion GUS gene between Hind III and BamH I of the pCAMBIAP1391Z vector as described in our previous study (Yan et al., 2016). Tobacco transformation was performed as described by Horsch et al. (1985), and the resulting transgenic tobacco plants were selected and self-pollinated to obtain homozygous T2 offspring, which were used for all subsequent analyses. Plants were grown in pots (15 cm diameter × 13 cm height) filled with a mixture of nutrient soil, perlite, and vermiculite (1:1:1, v/v/v) and watered with sterile water in the greenhouse. Greenhouse conditions included a long-day photoperiod (14 h light, 10 h dark), 25 to 28 °C, 2000 lx, and 70% relative humidity.
Flowering time analysis.
Beginning on the first day of flowering, the number of flowers was recorded every 2 d until the full-bloom stage on 30 plants each of the wild-type and DRL1 transgenic (L1, L3) lines. Data are reported as averages for each genotype.
Gus histochemical analysis.
For histochemical staining of GUS activity, samples were vacuum infiltrated with GUS staining solution for 30 min (Roni et al., 2006; Zhu et al., 2019). A tobacco flower develops over 12 d from a flower bud to a fully bloomed flower. Five different stages of floral development (f1, f2, f3, f4, and f5) of wild-type and DRL1 transgenic tobacco are shown in Fig. 1. Flowers from each stage were stained with GUS, and images of the staining patterns were collected with a microscope and digital camera [10 × 40 (Ds-Fi1C; Nikon, Tokyo, Japan)].
GUS staining patterns in wild-type and DRL1pro::GUS tobacco plants. (A) Five different stages of floral development in wild-type tobacco. (B) GUS staining of wild-type tobacco at different developmental stages. (C) Five different stages of floral development in transgenic tobacco. (D) GUS staining of transgenic tobacco at different developmental stages. (E–I) Magnified images of GUS staining in transgenic tobacco at different developmental stages.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 1; 10.21273/JASHS04968-20
Hybridization of transgenic plants and observation of fruit characteristics.
Pollen viability determination.
Statistical analysis.
All values were expressed as the mean ± sd of three replicates. Data were analyzed by one-way analysis of variance, and differences between wild-type and individual transgenic lines were considered significant at P < 0.05. Analyses were performed with statistical software (SPSS version 19.0 for Windows; IBM Corp., Armonk, NY).
Results
Effect of DRL1 overexpression on tobacco flowering time.
Wild-type tobacco began to bloom 36 d after planting, whereas DRL1 transgenic tobacco lines L1 and L3 began to bloom 58 d after planting (Fig. 2). The number of flowers produced by the L1 and L3 transgenic tobacco lines did not differ significantly from that of wild-type tobacco (P < 0.05). These results suggest that DRL1 overexpression delayed tobacco flowering time but did not affect flower number.
Flowering times of wild-type and DRL1 transgenic L1 and L3 tobacco. Data are presented as mean ± sd of three independent experiments.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 1; 10.21273/JASHS04968-20
Analysis of DRL1 overexpression patterns by gus staining.
Figure 1 shows GUS activity in wild-type and DRL1pro::GUS transgenic tobacco plants. GUS staining was detected in sepals and petals at the first and second stages of floral development (f1 and f2). A reduction in GUS staining was observed in sepals and petals from f3 to f5, and GUS activity was almost undetectable in later developmental stages. GUS staining was detected in pistils and stamens throughout floral development and increased from f1 to f5 (Fig. 1D). Magnified images of the five floral development stages (Fig. 1E–I) revealed strong GUS staining in stigmas and anthers, indicating that DRL1 was highly expressed in these tissues.
Effect of DRL1 overexpression on tobacco fruit.
Table 1 showed that S1 had the highest fruit set rate of 96.7% ± 0.04%. The average fruit set rate of C1 was only 25.6% ± 0.03% and was significantly lower than that of S1 (P < 0.05). Therefore, a transgenic male parent significantly reduced fruit set. The average fruit set rate of C2 was 91.1% ± 0.03%. The average fruit set rate of S2 was only 27.8% ± 0.03% and was significantly lower than that of C2 (P < 0.05). Therefore, a transgenic female parent had a minimal effect on fruit set. This result is further confirmed by the fact that the fruit set rate of S1 (96.7% ± 0.04%) did not differ significantly from that of C2 (91.1% ± 0.03%) (P < 0.05).
Fruit set of different hybridized combinations of wild-type and DRL1 transgenic tobacco.
Differences in fruit weight among crosses followed the same pattern as that observed for fruit set. The fruit weight of S1 was the highest (0.497 ± 0.03 g). The average fruit weight of C1 was only 0.227 ± 0.02 g and was significantly lower than that of S1 (P < 0.05). Therefore, a transgenic male parent significantly reduced fruit weight. The average fruit weight of C2 was 0.473 ± 0.02 g. The average fruit weight of S2 was only 0.256 ± 0.02 g and was significantly lower than that of C2 (P < 0.05). Therefore, a transgenic female parent had a minimal effect on fruit weight. The same patterns were observed for seed weight, fruit shape indice, and seed germination rate. Furthermore, the cross with a transgenic female parent exhibited normal fruit development and morphology (Fig. 3), but the fruit of the cross with a transgenic male parent were dry and long, with abnormal development. DRL1 overexpression appeared to have a marked effect on anther and/or pollen function but little effect on female reproductive activity.
Fruit set of different genotype crosses of tobacco. (A–D) Phenotypic observations of fruit set in the WT (♀) × DRL1 (♂) cross. The fruit were dry and long and exhibited abnormal development. (E–H) Phenotypic observations of fruit set in the DRL1 (♀) × WT (♂) cross. The fruit exhibited normal development. Red arrows in (A) and (E) indicate developing fruit.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 1; 10.21273/JASHS04968-20
Effect of DRL1 overexpression on tobacco pollen vigor.
Pollen vigor was measured to further assess pollen function. Table 2 shows that the in vitro germination percentage of wild-type pollen (86.84% ± 0.06%) was significantly greater than that of transgenic pollen (14.08% ± 0.04%) (P < 0.05). Indeed, little of the transgenic pollen germinated (Fig. 4A and B). We further analyzed the effect of DRL1 overexpression on tobacco pollen vigor using FDA staining (Fig. 4C and D). Over 92% of the wild-type pollen was viable, whereas only 25.00% ± 0.03% of the transgenic pollen was viable, and this difference was significant (P < 0.05).
Pollen viability of wild-type and DRL1 transgenic tobacco.
Pollen viability of DRL1 transgenic tobacco (A, C) and wild-type tobacco (B, D) was assessed by an in vitro germination test (A–B) and FDA staining (C–D).
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 1; 10.21273/JASHS04968-20
Taken together, our results suggest that the pollen produced by transgenic plants was much less active and viable than that of wild-type plants, consistent with the differences in fruit set and other reproductive parameters described above.
Discussion
About 3500 genes are specifically expressed during the development of anthers in higher plants, 60–100 of which are thought to play key roles in the control of anther development (Sanders et al., 1999). Characterizing the expression of key regulatory genes in anther and pollen development is therefore important to fully understand the molecular mechanisms of pollen development.
The NAC transcription factors (e.g., NTL8, LOV1, OsNTL5, NST, NAP, ATAF1, ATAF2, CUC2, and ANAC092) constitute a plant-specific transcription factor family with more than 100 members in many terrestrial plant genomes. The NACs are one of the largest transcription factor families in plants and are involved in all stages of plant growth and development, including the development of floral organs. For example, the overexpression of NTL8 in A. thaliana slows plant growth and delays flowering (Kim et al., 2007), whereas the overexpression of LOV1 in A. thaliana causes early flowering (Yoo et al., 2007). OsNTL5 overexpression in rice (Oryza sativa) significantly delays flowering and downregulates the expression of the flowering time regulators Ehd1, Hd3a, and RFT1. Chromatin immunoprecipitation with sequencing (ChIP-seq) results suggest that OsNTL5 binds to the CACG cis-element of the Ehd1 promoter, thereby delaying flowering time through the inhibition of Ehd1 expression. Other NACs have been shown to bind directly to the promoter elements of flowering pathway genes to influence flowering (Li et al., 2010). In the present study, the NAC transcription factor DRL1 from V. vinifera ‘Yatomi Rosa’ delayed the flowering time of DRL1 transgenic tobacco plants, consistent with previous studies of NAC genes and flowering time.
NAC transcription factors play important roles in stamen tearing and pollen loosening. For example, the A. thaliana NAC transcription factors NST1 and NST2 regulate secondary wall synthesis in pollen sac endothelial cells, thereby controlling stamen rupture (Mitsuda et al., 2005). MYB26 from A. thaliana controls stamen secondary wall thickening through its regulation of NST1 and NST2. Likewise, a Tnt1 retrotransposon insertion inhibits alfalfa (Medicago sativa) pollen sac cleavage in an mtnst1 mutant due to thickening of the secondary wall (Yang et al., 2017). The A. thaliana NAC gene ANAC092 is also expressed during specific periods of anther and pollen development (Li et al., 2013). In the present study, DRL1pro::GUS transgenic tobacco showed the strongest GUS staining in stigmas and anthers. As floral development progressed, stigma and anther staining gradually darkened, indicating that the expression of DRL1 gradually increased during the development of these organs.
Arabidopsis thaliana NARS1/NAC2 and NARS2/NAM belong to the NAC subfamily III. Double mutants exhibit seed shrinkage, but single mutants show no phenotype, indicating that there is functional redundancy between NARS1 and NARS2. In crosses between wild-type and nars1 nars2 double mutants, normal seeds formed when the mutant parent was male, but abnormal seeds formed when the mutant parent was female, suggesting that NARS1 and NARS2 affect ovule development (Kunieda et al., 2008). In the present study, overexpression of DRL1 in tobacco resulted in reduced pollen viability and germination. Likewise, crosses in which DRL1 tobacco was the male parent produced a low fruit set and small fruit, and these effects were not present when the transgenic parent was female. DRL1 appeared to have a significant effect on anther function but a marginal effect on stigma function.
NAC transcription factors are unique to plants, and their functional characterization is a topic of current interest. Their functions are wide-ranging and include the regulation of cell division, flower development, plant senescence, secondary wall growth, lateral root development, stress resistance, and disease resistance (Guo et al., 2018; Hu et al., 2010; Nuruzzaman et al., 2010; Zhong and Ye, 2015). This study focused on changes in flowering time, floral development, and fruit set when the NAC transcription factor DRL1 from ‘Yatomi Rosa’ grape was overexpressed in tobacco. The specific expression patterns we observed were consistent with a role for DRL1 in the morphogenesis of floral organs in grapevine. Future work will focus on DRL1’s effects on lateral root growth, ovule development, disease resistance, and stress resistance, further enriching our theoretical understanding of the regulatory mechanisms of the NAC transcription factors.
Conclusions
Our results demonstrate that DLR1 overexpression strongly influences flowering time and reproduction in transgenic tobacco, primarily through its effects on pollen development. Given the central role played by NAC transcription factors in plant growth and development, the present work will be of interest to grape researchers for the development of improved germplasm, as well as to researchers with an interest in the function of NAC family transcription factors.
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