miR319-Regulated TCP3 Modulates Silique Development Associated with Seed Shattering in Brassicaceae

Seed shattering is an undesirable trait that leads to crop yield loss. Improving silique resistance to shattering is critical for grain and oil crops. In this study, we found that miR319-targeted TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCPs) inhibited the process of post-fertilized fruits (silique) elongation and dehiscence via regulation of FRUITFULL (FUL) expression in Arabidopsis thaliana and Brassica napus. AtMIR319a activation resulted in a longer silique with thickened and lignified replum, whereas overexpression of an miR319a-resistant version of AtTCP3 (mTCP3) led to a short silique with narrow and less lignified replum. Further genetic and expressional analysis suggested that FUL acted downstream of TCP3 to negatively regulate silique development. Moreover, hyper-activation of BnTCP3.A8, a B. napus homolog of AtTCP3, in rapeseed resulted in an enhanced silique resistance to shattering due to attenuated replum development. Taken together, our findings advance our knowledge of TCP-regulated silique development and provide a potential target for genetic manipulation to reduce silique shattering in Brassica crops.


Introduction
Angiosperms show various types of siliques, most of which are derived from the ovary walls and fertilized ovules. Their structures are conserved in Arabidopsis thaliana and some species of the Brassicaceae family, including cabbage, broccoli, Chinese cabbage (Brassica rapa), and rapeseed (Brassica napus) [1]. In Arabidopsis thaliana, siliques are composed of fertilized ovules and three major regions, namely the valves, repla, and valve margins. The valve margins were formed between the valves and the repla, and two types of cells at the valve margins regulated the silique opening. The separation layer is also called dehiscence zone (DZ), which is composed of 2-3 layers of thin-walled cells and separates the heavily lignified cells of the pericarp edge from the replum. Fruit dehiscence is accompanied by degradation of the thinwalled cells [2][3][4][5]. Canola seeds, which are harvested for oil, are often lost owing to premature silique shattering, particularly under adverse weather conditions, and premature dehiscence or silique shattering leads to significant crop losses [6]. Reducing silique shattering will increase the proportion of seed obtained during harvest, which is conducive to economic income.

Plasmid Construction and Transformation
To generate the mAtTCP3-overexpression construct, the full-length CDS of AtTCP3 (AT1G53230) was amplified using specific primers. Then, a silent mutation was introduced into the miR319 target site of the AtTCP3 CDS to generate the mAtTCP3 construct using a Site-Directed Mutagenesis Kit (TaKaRa). The chimeric pAtTCP3::AtTCP3SRDX was constructed as described previously [32]. To generate the p35S::AtFUL construct, the full length CDS of the AtFUL gene was amplified. To generate the p35S::BnTCP3.A8 construct, the full length CDS of the Brassica napus TCP3 gene (Acc. number GSBRNA2T00114181001) was amplified and cloned into the binary vector pChimera. The Agrobacterium tumefaciens strain GV3101 pMP90RK was used for stable plant transformations. All the primers used for plasmid construction are listed in Table S1.
For rapeseed hypocotyl transformations, we followed the protocol described by Liu et al. and Moloney et al. [33,34]. The p35S::mAtTCP3, pAtTCP3:: AtTCP3SRDX and p35S:: AtFUL vectors were transferred into Col-0 using the floral dip method [35]. The p35S::BnTCP3.A8 and p35S::mAtTCP3 transgenic plants were selected on MS medium containing 50 mg/mL kanamycin after Agrobacterium-mediated transformation. Positive seedlings (T0) were transplanted into soils. In the F1 generation, all transgenic plants were confirmed using PCR with gene-specific primers and maintained in a growth room at 22 • C under long-day conditions. Primers used in this study are listed in Supplementary Table S1.

Isolation of RNA and Real-Time PCR Analysis
Total RNA was extracted from plants using TRIzol reagent. Before performing quantitative real-time PCR, RNA was treated with DNase I (TaKaRa) to avoid DNA contamination, and then RNA was purified with the phenol chloroform. Real-time detection of all target genes was based on the method of Li et al. (2020) [36]. The quantification of the relative expression levels was performed as reported previously [37]. The relative transcript levels of each gene in Arabidopsis thaliana and B. napus were normalized to that of Actin [36] and UBC21 [38] for quantitation, respectively. Primer Premier 5 software was used to design oligonucleotide primers (http://www.premibiosoft.com (accessed on 1 September 2022)) for real-time PCR to amplify the reference and target genes. All the primers used in this study are shown in Table S1.

Histological Analyses
For toluidine blue staining, the siliques were harvested one week after opening of flower buds and fixed in FAA for more than 18 h, sectioned and stained with 0.1% toluidine blue [7,8,39]. For phloroglucinol lignin staining, the siliques at the same stage were stained with 2% phloroglucinol solution overnight. Then, the siliques were decolorized in ethanol and photographed.

In Situ Hybridization
Silique (stage [10][11][12] sections in the wild-type flowers were prepared using previously described pretreatment and hybridization methods [40]. The primers used to generate hybridization probes specific for AtTCP3 using the fragments (513 bp) in coding sequences according to the method of Wang et al. (2015) [30]. LNA (Locked Nucleic Acid) modified probes specific for AtTCP3 were synthesized and labeled with DIG at the 3 -end by TaKaRa.

Scanning Electron Microscopy (SEM)
Siliques (stage 17) of Arabidopsis and B. napus were harvested and fixed overnight at 25 • C in FAA, dehydrated through an ethanol series, and critical-point dried. Samples were sputter-coated with gold and viewed using a Hitachi S-2300 electron microscope. Replum

Shattering-Resistance Measurements
Mature siliques were collected for the measurement of silique shattering resistance by a random impact test (RIT) [41]. In total, 20 siliques were preserved in a mesh bag overnight after incubation at 60 • C for 60 min to equilibrate the moisture content, and then, they were placed into a cylindrical container, having a diameter of 6 cm and a height of 17 cm, which was pre-loaded with 12 steel balls with a diameter of 13 mm. On a horizontal shaker, the container was shaken at 300 rpm and then the numbers of cracked siliques were recorded after 1 min shaking intervals. A total of 10 recordings were taken. After each recording, the broken siliques were removed, and all the experiments were performed in triplicate. The silique shattering-resistance index (SRI) was calculated using the following equation: where Xi represents the number of cracked siliques in time i th , with 1 ≤ i ≤ 10.

Sequence alignment and Phylogenetic Analysis
Arabidopsis AtTCP protein sequences were downloaded from the Arabidopsis Information Resource (https://www.arabidopsis.org/ (accessed on 1 September 2022)). BnTCP3.A8 and Brp.TCP3 were selected based on their high similarity with AtTCP3. Full-length amino acid sequence multiple alignments were performed using ClustalW and GeneDoc. Unrooted phylogenetic trees were constructed from the aligned amino acid sequences using the neighbor-joining method in MEGA 6.0, and bootstrapping was carried out with 1000 iterations [42].

Statistical Analysis
We calculate statistical significance using two-tailed Student's t-test and error bars indicate standard error (SE). Values of p < 0.05 are statistically significant.

AtMIR319a-Regulated TCPs Played Negative Role in Silique Development
In previous studies, the development defect of jaw-D mutant plant with activation of AtMIR319a was observed in both vegetable and reproductive stages . However, the molecular mechanism of silique defect in jaw-D plant has not been uncovered. Using scanning electron microscopy (SEM), we found that the repla of the jaw-D silique was wider than those of the wild type in the base, middle, and tip of the mature silique ( Figure 1A). Transverse sections of siliques showed that the jaw-D repla were significantly wider compared with wild-type repla (Col-0) in the middle of mature siliques ( Figure 1B,C). Thus, these results indicated that activation of AtMIR319a enlarged repla in siliques.
To investigate the expression changes of the five miR319-targeted TCP genes in different tissues of jaw-D mutant plants, real-time PCRs were performed to analyze the expression levels of AtTCP2, AtTCP3, AtTCP4, AtTCP10, and AtTCP24 in cauline leaves, inflorescences, flowers, and siliques. In cauline leaves and inflorescences, the expression of AtTCP2, AtTCP3, AtTCP4, AtTCP10, and AtTCP24 were consistently reduced to a similar level in jaw-D mutants compared to the wild type ( Figure 2A). In contrast, their expressions were differently suppressed in flowers (stage 14-15) and siliques (stage 17). Specifically, AtTCP3 was mostly suppressed among all the five TCP genes in silique of jaw-D plants (Figure 2A). Detailed analysis of AtTCP3 expressional level in different plant tissues confirmed that this gene was most accumulated in siliques as compared to in rosette leaves, inflorescences, and flowers ( Figure 2B). Given the siliques of Arabidopsis thaliana originated from gynoecium with two fused carpels, we further investigated the mRNA abundance of AtTCP3 during early flowering stage by in situ hybridization. In young flower buds of wild-type plants, AtTCP3 transcripts were highly accumulated in the developing carpel ( Figure 2C). These results suggested that AtTCP3 was one of the major regulators of silique development among these five miR319 targeted TCPs.

AtTCP3 Negatively Regulated Repla Development and Reduced the Lignification in Siliques
To further study the function of TCP3 in developing siliques, a miR319a-resistant version (p35S::mAtTCP3) of AtTCP3 was introduced into Arabidopsis. As expected, the AtTCP3 transcription level was significantly increased in the transgenic plants compared with wild-type ( Figure 3A). In wild-type siliques, valve margins or DZ were differentiated as a narrow strip consisted of few cells within the separation layer and the lignified regions, both of which contributed to the active fruit-opening process [19]. Consistent with Notably, TCP4 and TCP24 were also significantly repressed in silique of jaw-D plants compared to Col-0 ( Figure 2A). Knockout mutants of AtTCP3 showed no visible phenotypic alterations due to the functional redundancy of the miR319-target TCPs [43,44]. To further confirm the function of AtTCP3 in repla development, we constructed the pAtTCP3::AtTCP3SRDX transgenic line fusing TCP3 with plant-specific ERF-associated amphiphilic repression (EAR) motif repression domain (SRDX), which suppressed target genes of endogenous TCP3 and its functionally redundant TCPs. We found that the replum width of the pAtTCP3::AtTCP3SRDX siliques was similar to jaw-D siliques ( Figure 1C). These results further suggested that miR319-regulated TCPs played critical role in replum development in siliques.

AtTCP3 Negatively Regulated Repla Development and Reduced the Lignification in Siliques
To further study the function of TCP3 in developing siliques, a miR319a-resistant version (p35S::mAtTCP3) of AtTCP3 was introduced into Arabidopsis. As expected, the AtTCP3 transcription level was significantly increased in the transgenic plants compared with wild-type ( Figure 3A). In wild-type siliques, valve margins or DZ were differentiated as a narrow strip consisted of few cells within the separation layer and the lignified regions, both of which contributed to the active fruit-opening process [19]. Consistent with the previous studies [43], p35S:mAtTCP3 plants were smaller than wild-type plants ( Figure 3B), and their siliques were much shorter ( Figure 3C-E). Based on the SEM image of siliques (Stage 17), the repla of p35S:mAtTCP3 siliques were much narrower than those in wide type and jaw-D siliques ( Figure 1A). Cross sections in the middle of siliques also showed that the replum cells were relatively smaller in p35S:mAtTCP3 siliques than in wild type and jaw-D. In addition, a layer of large and sparse cells were seen between the valve and the replum in the Col silique, which facilitated the dehiscence of the siliques, whereas this layer of cells was much denser in p35S::mAtTCP3 siliques ( Figure 1B). These results suggested that AtMIR319a-regulated TCP3 functioned as a negative regulator of silique development. the previous studies [43], p35S:mAtTCP3 plants were smaller than wild-type plants (Figure 3B), and their siliques were much shorter ( Figure 3C-E). Based on the SEM image of siliques (Stage 17), the repla of p35S:mAtTCP3 siliques were much narrower than those in wide type and jaw-D siliques ( Figure 1A). Cross sections in the middle of siliques also showed that the replum cells were relatively smaller in p35S:mAtTCP3 siliques than in wild type and jaw-D. In addition, a layer of large and sparse cells were seen between the valve and the replum in the Col silique, which facilitated the dehiscence of the siliques, whereas this layer of cells was much denser in p35S::mAtTCP3 siliques ( Figure 1B). These results suggested that AtMIR319a-regulated TCP3 functioned as a negative regulator of silique development.  Silique wall thickness and replum development are correlated with lignin accumulation in plants [5]. To investigate whether TCP3 play a role in regulating replum lignification, phloroglucinol, a lignin-specific histological stain, was applied to siliques of wild-type, p35S::mAtTCP3, pAtTCP3::AtTCP3SRDX, and jaw-D plants for evaluating the degree of tissue lignification. In the wild-type siliques, lignin-specific signals were clearly seen in the repla and valve margin cells adjacent to the DZ throughout the siliques, while the signals were very faint in the repla and valve margin cells of p35S::mAtTCP3 siliques ( Figure 3F). In contrast, the lignin-specific signals were much stronger in the repla and valve margin cells of jaw-D and pAtTCP3::AtTCP3SRDX siliques compared to those in wide type. These results indicated that AtTCP3 overexpression attenuated lignification of repla in the siliques.

AtFUL Acted Downstream of AtTCP3 in Replum Deficiency
Recent molecular and genetic studies in Arabidopsis have identified several crucial genes involved in the regulation of silique development [45]. To determine the relationship of TCP3 with those valve-and valve-margin-related genes, the expressional level of AtFUL, AtNTT, AtIND, AtALC, AtJAG, AtFIL, AtYAB3, and the replum-related gene AtPRL, were analyzed using real-time PCR. Transcript levels of AtFUL and AtRPL were significantly elevated in the p35S::mAtTCP3 plants as compared to wild-type ( Figure 4A). In addition, transcript levels of AtFUL, AtIND, AtRPL, and AtJAG were significantly downregulated in the jaw-D mutants compared to those in the wild type ( Figure 4A). In combination, these results implied that AtTCP3 acted as an upstream regulator of AtFUL to suppress the differentiation of the replum.
To further understand the genetic relationship between AtTCP3 and AtFUL, the AtFUL gene were either overexpressed or suppressed in jaw-D mutant background. In FUL jaw-D plant overexpressing FUL, silique repla were narrower in width than that of jaw-D silique but still wider than the wild type, indicating that the wide replum phenotype of jaw-D mutant was partially complemented by AtFUL (Figures 1C and 4B). Besides, the wavy leaf margins phenotypes observed in jaw-D were also restored by AtFUL overexpression in FUL jaw-D plants ( Figure 4C). Moreover, ful jaw-D double mutants showed similar repla width with ful mutant (Figures 1C and 4D). Together, these results suggest that AtFUL acted downstream of TCP genes.

Hyper-Activation of BnTCP3.A8 Affected Silique Development in Rapeseed
Rapeseed and Arabidopsis are members of the Brassicaceae family and display similar silique morphologies. To investigate whether TCP3 is also involved in replum development of rapeseed, we identified the B. napus TCP3 gene by blast searching for the AtTCP3 protein coding sequence in the Brassica database (BRAD). The obtained locus, GSBRNA2T00114181001, which located on chromosome A08, showed the highest similarity to AtTCP3. Thus, this gene was named as BnTCP3.A8 in following studies ( Figure 5A, Figure S1A,B). Further sequence alignment of this gene in the same database revealed that BnTCP3.A8 displayed a 98% similarity to B. rapa BrpTCP3 ( Figure 5A). BnTCP3.A8 also contained a conserved BrpmiR319a-targeted site within the identified sequences ( Figure 5B).

Hyper-Activation of BnTCP3.A8 Affected Silique Development in Rapeseed
Rapeseed and Arabidopsis are members of the Brassicaceae family and display similar silique morphologies. To investigate whether TCP3 is also involved in replum development of rapeseed, we identified the B. napus TCP3 gene by blast searching for the AtTCP3 protein coding sequence in the Brassica database (BRAD). The obtained locus, GSBRNA2T00114181001, which located on chromosome A08, showed the highest similarity to AtTCP3. Thus, this gene was named as BnTCP3.A8 in following studies ( Figure 5A, investigate whether the p35S::BnTCP3.A8 plants also reduced lignification in repla, the siliques of both the wild-type and transgenic lines were stained by phloroglucinol. The siliques of T26 showed weaker lignin-specific signals in the repla than those of the wildtype ( Figure 6H). In addition, the siliques of T26 exhibited smaller valve margin cells adjacent to the DZs ( Figure 6I). These observations indicated that the reduced lignification in the repla was responsible for the resistance of p35S::BnTCP3.A8 plants to silique shattering.  To study the functions of BnTCP3.A8 in silique development, the BnTCP3.A8 CDS was introduced into rapeseed plants (accession K407) under the regulation of the cauliflower mosaic virus 35S promoter. The expression of BnTCP3.A8 was significantly upregulated in p35S::BnTCP3.A8 transgenic lines T2, T4 (T2 generation), and T26 (T3 generation) ( Figure 6A,B). In those plants, silique lengths of the p35S::BnTCP3.A8 line were shorter than that of the wild-type ( Figure 6C,D). For immature gynoecium [46], the placenta development of p35S::BnTCP3.A8 line was weaker than that of the wild-type ( Figure 6E). Further, the image of SEM showed that the replum in T26 silique was much narrower than that of the wild-type ( Figure 6F). We measured the replum width in the middle of siliques. Replum width of T26 silique was significantly narrower than that of the wild-type ( Figure 6G). These results indicated that BnTCP3.A8 negatively regulated replum development in rapeseed, consistent with the role of AtTCP3 in Arabidopsis. Spence et al. (1996) showed that silique shattering resistance was negatively associated with the degree of lignification in the valves, repla, and valve margin cells [18]. To investigate whether the p35S::BnTCP3.A8 plants also reduced lignification in repla, the siliques of both the wild-type and transgenic lines were stained by phloroglucinol. The siliques of T26 showed weaker lignin-specific signals in the repla than those of the wild-type ( Figure 6H). In addition, the siliques of T26 exhibited smaller valve margin cells adjacent to the DZs ( Figure 6I). These observations indicated that the reduced lignification in the repla was responsible for the resistance of p35S::BnTCP3.A8 plants to silique shattering.
To define whether the deficiency in replum width of rapeseed affects silique shattering, we determined the silique shattering ratios using the modified method of Bruce et al. (2002) [47]. The siliques of the wild-type plants were opened easily by shattering treatment and released many seeds ( Figure 6K); however, the siliques of T26 plants were less opened than the wild-type and released fewer seeds. The SRI (shattering-resistance index) of T26 was 0.58 at 300 rpm, which was significantly high than the SRI of wild-type (SRI = 0.48) ( Figure 6L). Thus, these results suggested that overexpression of BnTCP3.A8 enhanced silique shattering resistance.

miR319-Targeted TCP Genes Inhibited Replum Enlargement
The miR319-targeted TCP genes control cell division arrest [25,27,28,[48][49][50]. In general, proteins encoded by genes expressed in the replum often negatively regulate genes expressed in the valves [1]. We found that these TCP genes were involved in development of repla because repla became wider in jaw-D mutant. Among them, AtTCP3 was downregulated mostly among these TCP genes. In situ hybridization revealed that AtTCP3 was preferentially expressed in middle region of carpel. In addition, p35S::mAtTCP3 reduced the replum width, and pTCP3::mAtTCP3SRDX increased the replum width. Taken together, these results suggested that AtTCP3 functioned in regulation of replum development.
The factors that control the establishment of medio-lateral silique patterns also regulate proper shoot development and leaf formation [51][52][53][54][55]. We found that AtTCP3 positively regulated valve-and valve margin-related genes AtFUL and AtSHP1. Genetic analysis showed AtFUL acted downstream of miR319a-targeted AtTCP3 in replum deficiency. This evidence indicates that TCP3 functioned in repressing replum enlargement.

BnTCP3.A8 Enhanced Silique Shattering Resistance
Cell wall lignification is a complex process that only occurs in higher plants, and its main function is to strengthen plant vascular bodies. Cell wall lignification affects silique dehiscence in siliques. SHP1 and SHP2 promotes valve margin lignification [8]. In this study, we found that the lignin-specific staining of the repla of Arabidopsis p35S::mAtTCP3 and p35S::BnTCP3.A8 plants was weaker as compared to wild-type, while the lignin-specific staining of the repla of jaw-D, and pAtTCP3::AtTCP3SRDX siliques were much stronger than that of the wild type.
The premature cracking of siliques before or during ripening causes silique shattering and drastically reduces production. Rapeseed is widely planted in temperate regions. Total yields may reduce 20% owing to silique shattering, and in arid environments this reduction reaches 50% [56,57]. In recent years, some progress has been made in transgenic approaches using Arabidopsis genes to produce indehiscence rape [58]. Chung et al. (2013) reported that the ntt-3D mutant, an activation tagged allele of NTT, showed an enlarged replum and the fruit indehiscence in Arabidopsis [13]. Overexpression of AtFUL gene in B. juncea produces pod shatter-resistant Brassica fruit [19]. The shatter-resistant B. juncea siliques may have smaller non-lignified separation layers than B. napus siliques [59]. A similar phenotype was observed in 35S::FUL Arabidopsis plants, which showed reductions in the lignification of cells adjacent to the DZs, resulting in the formation of indehiscent siliques that did not release seeds normally [20]. The replum of the transgenic line p35S::BnTCP3.A8 displayed a narrower replum and a lower degree of lignification, compared with that of the wild type, and t p35S::BnTCP3.A8 plants exhibited higher silique shatter resistance. Thus, the deficiency in replum may affect silique shattering. Whether are the size and shape of valve margin changed by TCP3 deregulation remains unclear. Both the Arabidopsis p35S::mAtTCP3 and B. napus p35S::BnTCP3.A8 plants exhibited shorter and smaller siliques. This fact implies that the effects of TCP3 on silique development are much broader than expected. An attempt to explore the new function of TCP3 and the other miR319a-targeted gene is underway in our lab. In FUL jaw-D double mutants, silique repla were narrower in width than that of jaw-D silique but still wider than the wild-type, and ful jaw-D double mutants no significant change of repla width was observed compared to ful mutant. TCP3 positively regulated FUL expression and affected replum lignification in Arabidopsis and B. napus. Alternatively, TCP3 may inhibit replum growth in a direct or indirect path unknown. Our results provide novel insights into the mechanisms of silique dehiscence in Arabidopsis and B. napus, which can most likely be applied to other crops and lay a foundation for the development of oil crop varieties having strong shattering-resistance levels.

Conclusions
In Summary, this study revealed a critical role of miR319-regulated TCPs in silique development, reducing replum width and lignification via FUL-regulated pathway, which contributed to the silique shattering resistance in both Arabidopsis and B. napus. Specifically, we found that hyper-activation of BnTCP3.A8 enhanced silique resistance to shattering in rapeseed, providing a potential genetic locus for molecular breeders to improve silique shattering resistance in Brassica crops.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11193096/s1, Table S1. Primer and probe sequences used in this study; Figure S1. Alignment and phylogenetic analysis of miR319-regulated TCP proteins in