Early flowering of the transgenic rice with AtNF-YB1

doi:10.21203/rs.3.rs-2088846/v1

Grain yield and growth period are two critical agronomic traits for a rice cultivar to be used in the field. The growth period is determined by flowering time, which also greatly affects grain yield. An Arabidopsis gene, AtNF-YB1, was introduced into rice, including Kasalath and two hybrids, Jinfeng X Chenghui 727 and Jinfeng XChuanhui 907. All the transgenic rice showed early flowering in both natural long-day (NLD) and natural short-day (NSD) conditions because of suppression of Ghd7 transcription resulting in early activating the flowering transition. The of pure line of Kasalath with the gene showed also shorter plant height and less grain yield with a decrease in spike length and grain number but more productive panicle. But, for the hybrids, a little or no reduction of spike length, grain number, and more productive panicles were observed so that grain yields were kept or even increased under NLD conditions. RNA-Seq further demonstrated that three pathways related to plant photosynthesis were markedly up-regulated in the transgenic Jinfeng B and hybrid Jinfeng X Chuanhui 907. Measurements of photosynthetic parameters indicated that photosynthetic rates were up-regulated with an increase of chlorophyll contents in the transgenic Jinfeng B and hybrid Jinfeng XChuanghui 907. The results suggest that AtNF-YB1could be useful for creating rice variety with early ripening, particularly for hybrid rice.

Oryza sativa L.

AtNF-YB1

Hybrid rice

Flowering

Photosynthesis

Grain yield.

In rice expression of AtNF-YB1 activated flowering pathway by suppression of Ghd7 transcription. Meanwhile, it also enhanced carbon assimilation reducing the side effect of the early flowering on grain yield.

In crops, flowering time (or heading date) is a major factor in determining their adaptation to specific cultivation areas and particular seasons of a year, which subsequently affect the grain yields and even the grain qualities. Cultivated rice (Oryza sativa L.) is domesticated from its progenitor, wild rice (Oryza rufipogon Griff.), in the lower latitude areas. Rice is thus originally a facultative short-day (SD) plant, flowering during shorter summer days. However, rice as a staple crop is now widely cultivated globally from 55 °N to 36 °S flowering in both short- and long-day (LD) conditions (Khush 1997; Chen et al. 2019).

It is known that two florigens in rice, Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1), are synthesized in leaves and then moved to shoot meristem, inducing flowering transition (Kojima et al. 2002; Komiya et al. 2008; Komiya et al. 2009). Further, two integrators, Hd1 and Ehd1, regulate expressions of the two florigen genes in response to environmental signals, particularly the natural photoperiod of sunlight and the circadian clock (Yano et al. 2000; Doi et al. 2004; Wei et al. 2016). Hd1 is conserved with Arabidopsis gene CONSTANS (CO) in the regulation pathway of OsGI-Hd1-Hd3a/RFT1, in which OsGI has a rhythmic expression pattern coincident with diurnal changes stimulating Hd1 expression and further activating Hd3a expression (Kojima et al. 2002). But, Ehd1 is specific to rice and plays a central role in the pathway of Ehd1- Hd3a/RFT1 (Doi et al. 2004). Its expression is usually inhibited in the LD condition by Ghd7/ EH7/Hd4 (Xue et al. 2008), DTH8/Ghd8/ Hd5 (Wei et al. 2010b; Yan et al. 2011), and PRR37/DTH7/Ghd7.1/Hd2 (Koo et al. 2013; Gao et al. 2014; Yan et al. 2013). Interestingly, Hd1interacts with the suppressor genes, Ghd7 (Nemoto et al. 2016; Zhang et al. 2017), DTH8 (Du et al. 2017; Cai et al. 2019), and OsPRR37 (Goretti et al. 2017; Fujino et al. 2019), resulting in repression of the activities of Ehd1-Hd3a/RFT1 pathway in LD conditions. Besides, the other upstream genes of Ehd 1, such as OsNTL5 (Guo et al. 2018), and DHD1 (Zhang et al. 2019) also function as a transcriptional repressor to suppress rice flowering. On the contrary, Ehd1 can be activated by some genes like OsMDAS51 (Kim et al. 2007), Ehd2/RID1/OsID1 (Matsubara et al. 2008; Wu et al. 2008), Ehd3 (Matsubara et al. 2011), Ehd4 (Gao et al. 2013), and OsMADS50 (Lee et al. 2004) under SD or LD conditions or both. In addition, DTH2 directly activates Hd3a/RFT1expression to promote flowering under LD condition (Wu et al. 2013), but Se14 directly inhibit RFT1expression through demethylation of H3K4me3 of RFT1 to repress the flowering transition under LD condition (Yokoo et al. 2014).

Variations of the so-called flowering-time gene (or heading-time gene), involved in regulating the florigen gene expressions, certainly result in changes in flowering time (Takahashi et al. 2009; Ogiso-Tanaka et al. 2013). Loss or attenuated function of the repressors such as Ghd7, DTH8, PRR37, and Hd1, allows rice to flower under LD and be cultivated in higher latitude areas (Zhang et al. 2015; Zhang et al. 2021). The cultivars fitted to such environment show photoperiod insensitivity as both Hd3a and RFT1 are de-repressed, regardless of photoperiod (Gómez-Ariza et al. 2015). However, loss-of-function genes of Ghd7, GTH8, PRR37, or Hd1 are also popular among rice cultivars in tropical and subtropical regions, as selected in modern breeding practice (Kim et al. 2018; Zhang et al. 2019; Zong et al. 2021).

Diversified flowering time makes rice adapt to various growing areas and cropping seasons. However, it also increases the complexity of breeding a variety with suitable growth duration for a specific region. Usually, breeders have to blindly make a lot of crosses for repeated testing in the field, resulting in low breeding efficiency and increased workload (Wei et al. 2010a; Hu et al. 2019). With current molecular tools such as transgenic or gene editing, it is feasible to precisely modify gene or gene expression based on known genetic information, making breeding more direct and efficient than the conventional method (Cui et al. 2020; Zhou et al. 2022).

In this study, we introduced an Arabidopsis gene AtNF-YB1 into rice Kasalath, and the transgenic plants showed early flowering under both NLD and NSD conditions. To further test the possibility of creating a variety with various growth periods in broad fields, we transferred the gene to a male sterile maintainer line, Jinfeng B, and subsequently to its male sterile line Jinfeng A via backcross approach. Finally, the transgenic male sterile line was crossed with restorer lines creating hybrids containing the transgene. The obtained transgenic hybrids showed early flowering, having potential usage in field production.

Gene expression cassette and transgenic plants

A 426 bp PCR fragment of AtNF-YB1 coding sequence amplified from Arabidopsis (NCBI Reference Sequence, NM_001336741.1) was cloned into a pCXUN vector (Chen et al. 2009) and verified by sequencing with primer pairs in Table S1. The obtained construct ubiquitin: :AtNF-YB1 was then transferred into Agrobacterium tumefaciens LBA4404 by the liquid nitrogen freeze-thaw method (An et al.1988). Two rice cultivars, Kasalath and Jinfeng B, were transformed with the Agrobacterium containing the plasmid (Saika and Toki 2010; Sahoo et al. 2011), and the regenerated positive plants in the media with hygromycin were selected on marker hptII and confirmed with expression of AtNF-YB1 in the plants. DNA was extracted from the leaves using the cetyltrimethyl ammonium bromide method (Doyle and Doyle 1987). PCR was conducted with the respective primers listed in table S1, following the protocol recommended in the manual of PCR reagents (Takara Dalian, China). Homozygous transgenic lines with a single copy of the transgene expressed were selected from T₂ segregating populations and further confirmed by PCR. To obtain hybrid rice with AtNF-YB1, the transgenic Jinfeng B was first crossed with the corresponding male sterile line Jinfeng A, and then the F1 plants were backcrossed with the transgenic Jinfeng B. In the following backcross population, homozygous male sterile lines with the transgene were identified. Finally, the male sterile line with the homozygous AtNF-YB1 gene crossed with two restorer lines of Chuanhui 907 and Chenghui 727, and hybrid seeds were produced and used in the field experiments. Expressions of AtNF-YB1 in the hybrids were examined using reverse transcriptase PCR with the gene-specific primers (Table S1).

Field experiments and phenotypic traits

For the NLD experiments, rice plants were grown in the paddy field from March to August at Pengzhou Experimental Station in Chengdu (31.0 °N, 104.1 °E), China. For the NSD test, rice was grown in the field from November to April at Yinzhou Experimental Station in Sanya (18.4 °N, 109.9 °E), China. The experiments were arranged in a completely randomized design with three replicates for each treatment. At harvest, five or more plants of each line were randomly chosen to measure yield traits. The grain number per panicle was measured as the total number of grains per plant divided by the number of panicles per plant. The 1000-grain weight (or 100-grain weight) was calculated as the weight of 1000 grains (or 100 grains) of the samples. Grain yield per plant (or 20-plant weight) was measured as the weight of total grains of one plant (or 20 plants). Excel 2010 was used for making charts and analyzing the data.

Real-time PCR and quantification of gene expressions

Total RNA was isolated from the leaf sample using Trizol reagent (Invitrogen, CA, USA). The cDNA was obtained using a PrimeScript First-Strand cDNA Synthesis Kit (Takara Dalian, China) following the protocol. The PCR amplification was conducted with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) according to the manufacturer’s instruction with specific primers (Table S1). The ubiquitin gene (LOC_Os03g13170) was used as an internal control. Relative expression levels of candidate genes were calculated using the 2^−ΔΔCt formula. The experiments were conducted with three or four replicates.

The flowering-time genes including Hd3a, RFT1, Hd1, Ehd1, GHd7, DTH8, and PRR37 were PCR-amplified from the genomic DNAs of Jinfeng B, Chenghui 727, and Chuanhui 907 and sequenced with the designed primers (Table S1). The genomic sequences of the seven genes in the three cultivars were deposited in GenBank under OP030513 to OP030533. The primers (Table S1) for quantifying the flowering-time genes in qRT-PCR were degenerated based on the sequences.

RNA sequencing and differentially expressed genes

Total RNA was isolated from young panicles (length, 5–10 mm) using the Trizol reagent (Invitrogen, CA, USA), and its concentration and purity were detected using Nanodrop2000. The integrity of the RNA was analyzed on agarose gel, and the RNA integrity number (RIN) value was further detected by Agilent 2100 (Agilent Technologies, USA). After that, mRNA was enriched by the oligo-dT selection, and cDNA was synthesized by reverse transcription. Then, the cDNA was ligated to the adaptor and further enriched by PCR amplification for 15 cycles. The DNA fragments were recovered from the target band on 2% agarose gel and then bridge amplified to form clonal clusters on cBot (Illumina, USA). Finally, the paired-end library was sequenced on the Illumina Hiseq X Ten sequencer with a read length of 2×150 bp. For each sequencing sample, at least six Gb raw data were generated. Three biological replicates were analyzed for each sample in the experiment. The raw data sets have been deposited in NCBI under accession PRJNA858181.

The obtained raw data were first filtered using Trimmomatic V. 0.39 (Bolger et al. 2014) to remove the adapter sequence and reads with low quality. The resultant clean data, with a minimum of 99% reads, achieved a quality score of Q30, and were then mapped to the rice reference genome MSU7.0 (Kawahara et al. 2013) using HISAT2 (Kim et al. 2105). On average, the reads mapping ratio for each sample was greater than 95%, and the mapped reads were further assembled using StringTie (Pertea et al. 2015). The expressions of genes was then quantified as Reads per Kilobase per Million Reads (RPKM), and differentially expressed genes were identified based on their expression using DESeq2 (Love et al. 2014). Criteria for differentially expressed genes (DEGs) were set as fold change (FC) ≥ 1.5 and FDR < 0.05.

Leaf chlorophyll and photosynthesis

The photosynthetic rate was measured in the morning from 9:00 to 11:00 A.M. The experiment was conducted using a Li-6400XT Portable Photosynthesis System (Li-Cor, USA). Fully expanded young leaves just on the heading stage were measured in a 2×3-cm leaf chamber with CO₂ 400 µmol s^-1and a built-in red + blue light-emitting diode light source of 1000 µmol m^-2 s^-1 at 28 °C. Ten or more plants were examined for each sample.

After measurement of the photosynthesis rate, the same leaves were sampled for chlorophyll analysis. Front part about 1/3 of the leaves were picked up. After removing the vein, the leaves were cut into small fragment and ground in the extraction solution (alcohol: acetone: water = 4.5: 4.5: 1). Leaf chlorophyll content was determined by measurement of absorbance at 645 and 663 nm using SpectraMax Plus 384 (Molecular Devices, USA). The pigment contents were calculated: Chlorophyll a (Chl a) =12.7 D663-2.59 D645; Chlorophyll b (Chl b) =22.9 D645-4.67 D663. Ten or more plants were examined for each sample.

AtNF-YB1 induces early flowering in rice

The gene, AtNF-YB1, was amplified by PCR method (The primers were listed in Table S1) using cDNA from Arabiopspsis (ecotype Columbia) leaves and cloned into plant expression vector pCXUN (Chen et al. 2009). AtNF-YB1 was first transferred into rice Kasalath. The plants in T₁ populations that contain the transgene detected in PCR showed early flowering; but the plants flowering lately as wild-type Kasalath were negative in the PCR examination. Further investigations were conducted using selected homozygous lines in the NLD condition (Chengdu, in summer) or NSD condition (Sanya, in winter). The transgenic Kasalath had fully flowered spikes with seed set; however, the wild-type was at its initial flowering stage (Fig. 1A). As a weak photoperiod-sensitive Indica landrace, Kasalath was flowering at about 105 days and 101 days in NLD and NSD, respectively. In contrast, the selected transgenic lines flowered early, at about 92 and 89 days in NLD and NSD, respectively.

To further explore the usage that AtNF-YB1 induces flowering early in breeding hybrid rice with shorter growth duration, the gene was transferred into rice Jinfeng B, a male sterile maintainer line of three-line hybrid rice. The gene was further introduced into Jinfeng A, the male sterile line, through backcrossing. As long as both the maintainer line and its sterile line containing AtNF-YB1as homozygote were ready, the two restorer lines, Chenghui 727 and Chuanhui 907, were respectively crossed with the sterile line to get two hybrids with the transgene, called transgenic Jinfeng X Chenghui 727 (T7) and transgenic Jinfeng X Chuanhui 907(T9).

The selected stable transgenic maintainer lines, Jinfeng B, and its sterile lines, Jinfeng A, were flowering early in the fields, about 12 days earlier under NLD conditions and four days under NSD. Both Jinfeng X Chenghui 727 (NT727) and Jinfeng X Chuanhui 907(NT907) were about 112 days to flowering under NLD. Under NSD, NT727 was 107 days and NT907 was 99 days to flowering. The transgenic hybrid T7 and T9 were both flowering at about 96 days after sowing under NLD in Chengdu. The transgenic plants were fully flowering (Figs. 1B and 1C), whereas the wild-type plants were at the flowering initial stages. Under NSD, the hybrids T7 and T9 were flowering at 95 and 88 days, respectively, in Sanya. All these indicate that the transgenic rice, either pure lines or hybrids, flower early under both NLD and NSD conditions.

AtNF-YB1 regulates shoot growth reducing plant height in rice

Although the height of the transgenic Kasalath plants seemed similar to that of the wild-type plants (Fig. 1A), height measurement at the flowering stage under NLD revealed a shorter phenotype of the transgenic plants (Fig. 1D). Despite few variations, the shorter statures of the transgenic lines were due to shorter length of each internode including the panicle (Fig. 1D). Shorter plant height among the transgenic lines was also observed during harvest under NLD (Table 1) and NSD (Table S2). The leaf numbers on the main culm were further investigated under NLD (Fig. 1E). Wild-type Kasalath had 12 or 13 leaves on the main culm with an average of 12.4, while the transgenic TK-01 had 11 or 12 leaves with an average of 11.3. These results indicate that AtNF-YB1 in Kasalath may result in reduced plant height with one less internode and shorter internode length of each internode, as well.

For hybrid rice, the transgenic plants of T7 and T9 were also shorter than its correspondent wild-type plants under NLD (Figs. 1F and 1G). However, the main panicle length was similar between transgenic and the correspondent wild-type plants for both hybrids. The first (I) and second (II) internode in the hybrids were even longer. Notably, the lengths of all the following internodes continually decreased. The final (VII) internode was almost invisible in both T7 and T9. Leaf numbers on the main culm in both T7 and T9 had about one less leaf than their correspondent wild-type hybrids (Fig. 1E). Under NSD, both hybrids with the transgene were shorter at harvest (Table S6). Therefore, expression of AtNF-YB1 in hybrid rice reduces plant height through regulation of the length of each internode; an internode is closer to the top, the length decrease is lesser. The top internode with panicle in the transgenic plants is even comparable to that in their wild-type plants.

AtNF-YB1 affects grain yield in rice

The transgenic Kasalath was cultivated in natural fields under NLD twice to investigate their agronomic traits (Table 1). Except for plant height, the main panicle length, the number of grains per panicle, and 1000-grain weight were reduced in the transgenic plants. However, the number of productive panicles per plant increased, decreased, or varied in different years or lines. The same phenomena were also observed under the NSD condition (Table S2). Thus, the transgenic Kasalath with AtNF-YB1 showed lower grain yields in both NLD and NSD conditions.

Several agronomic traits were investigated under NLD and NSD conditions for the two hybrids at harvest (Table 2). Plant heights were reduced; the main panicle lengths of transgenic and wild-type lines were similar in the mature plants under NLD conditions. The number of productive panicles was increased, but the 100-grain weight was reduced in the transgenic hybrids. The grain-setting rate between the transgenic and correspondent non-transgenic lines was similar, and the number of filled grains per panicle decreased in T7 but increased in T9. Thus, higher grain yields were recorded for the hybrids.

Under NSD conditions, the hybrids were tested twice in the winter season in Sanya. The reduced plant height with a shorter panicle was observed in both transgenic hybrids so that the filled grains on the panicle were reduced (Table S3). The 100-grain weight and the grain-setting rates decreased. However, the number of productive panicles per plant remained the same or increased. All of these resulted in a reduction of grain yields of both T7 and T9 in NSD.

AtNF-YB1 stimulates flowering pathways in rice

To explore the molecular basis of the early flowering, transcription levels of two florigens, Hd3a and RFT1, were measured first by a quantitative real-time PCR in Kasalath grown under NLD conditions. Both Hd3a (Fig. 2A) and RFT1 (Fig. 2B) were up-regulated in the transgenic line at the eighth week after sowing with peaks in the early morning or at midnight (Figs. 3A and 3B). The expressions of Hd1 (Figs. 2C and 3C) and Ehd1 (Figs. 2D and 3D), two major regulators of the florigen gene transcriptions (Sun et al. 2014), were induced in the sixth and seventh week before Hd3a and RFT1, respectively. Further analysis of the upstream genes in the flowering pathway revealed that Ghd7 (Figs. 2E and 3E) was clearly down-regulated before up-regulation of the florigen genes, but DTH8 (Figs. 2F and 3F) and PRR37 (Figs. 2G and 3G) had some up-or down-regulation after the flowering transition.

A diurnal expression experiment was then carried out under NSD conditions. Both Hd3a and RFT1 were up-regulated in the transgenic Kasalath (Figs. S1A and S1B). Hd1 and Ehd1 were also up-regulated (S1C and S1D). The expression of Ghd7 was decreased (Fig. S1E), but DTH8 and PRR37 were up- or down-regulated (Figs. S1F and S1G). These results are consistent with the observations under the NLD condition.

Hd1 in Kasalath genome is a non-functional allele (Yano et al. 2000). The observed up-regulation of Hd1transcriptions was thus not able to regulate the flowering induction. Ghd7, as only one among three upstream genes of Ghd7, PRR37, and DTH8 in the pathway of Ehd1-Hd3a/RFT1, was markedly down-regulated. Therefore, the suppression of the Ghd7 expression played a major role in promoting flowering in both NLD and NSD.

The genes in flowering-time pathways were also investigated in the two hybrids under NLD and NSD conditions. Under NLD, both T7 and T9 showed elevated expressions of both Hd3a (Figs. 2A and 3A) and RFT1 (Figs. 2B and 3B) at approximately the ninth week after sowing. The expressions of Hd1 (Fig.2C and 3C) and Ehd1 (Fig.2D and 3D) were also clearly up-regulated before the induction of the two florigen genes. Among the three upstream genes in the Ehd1- H3a/RFT1 pathway, Ghd7 (Figs. 2E and 3E) was the only dramatically down-regulated gene in the two transgenic hybrids before the up-regulation of the florigen genes. The transcript levels of DTH8 (Figs. 2F and 3F) and PRR37 (Figs. 2G and 3G) were only a little increased or decreased at the stage of flowering induction.

Under NSD conditions, expressions of Hd3a and RFT1 increased in both T7 and T9 (Fig. S1A, S1B). Ehd1 and Hd1 were also up-regulated (Fig. S1C, S1D). Ghd7 was dramatically down-regulated again (Fig. S1E). But, the transcript levels of DTH8 were unchanged (Fig. S1F), while PRR37 showed a little down-regulation (Fig. S1G). Thus, in the hybrids, suppressing the transcription of Ghd7 to activate the Ghd7-Ehd1-Hd3a/RFT1 pathway was also a major reason for the early flowering in both NLD and NSD conditions. However, Hd1 is a functional allele in Jinfeng B (GenBank, OP030521), so Hd1-Hd3a/RFT1 pathway could also impact the flowering induction in the hybrids.

AtNF-YB1 impacts multiple processes in rice

To further investigate the roles of AtNF-YB1 in rice, an RNA-seq experiment was carried out. Young spikes grow very fast after flowering induction in rice, playing a critical role in final grain yield. Therefore, spikes with a length of 0.5–1.0 cm were collected for the study. The experiment was designed to compare the transcriptions between the transgenic and corresponding wild-type plants, including Kasakath, the maintainer Jinfeng B, and two hybrids. The two hybrids were selected to be validated by quantitative real-time PCR with 16 genes (The primers were listed in Table S1). The PCR results were consistent with the results from RNA-Seq (Fig. S2).

Based on the criteria Fold Change (FC) ≥ 1.5 and P value < 0.01, 983 and 692 DEGs (Table S4) were identified in Kasalath (Table S5) and Jinfeng B (Table S6), respectively. In Kasalath, the up-regulated genes were in greater number than the down-regulated ones, whereas in Jinfeng B, the down-regulated genes were more than the up-regulated ones. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis showed that the top five regulated pathways in Kasalath were protein processing in the endoplasmic reticulum, endocytosis, galactose metabolism, spliceosome, and plant hormone signal transduction (Fig. 4A). For Jinfeng B, the pathway of protein processing in endoplasmic reticulum was also the most significantly enriched pathway as in Kasalath. Interestingly, three pathways, photosynthesis-antenna proteins, photosynthesis, and carbon fixation in photosynthetic organisms, appeared among the top five pathways in Jinfeng B (Fig. 4B). Looking into the transcript levels of the significantly regulated genes in the pathway of photosynthesis - antenna proteins, six among the seven genes were also up-regulated in Kasalath (Table S9). In the photosynthesis pathway, seven out of the 12 genes were expressed higher in Kasalath (Table S10). In the pathway of carbon fixation in photosynthetic organisms, four out of nine genes were up-regulated in Kasalath (Table S11).

In the two hybrids, 471 and 308 DEGs (Table S5) were respectively identified in Jinfeng X Chenghui 727 (Table S7) and Jinfeng X Chuanhui 907 (Table S8). Further analysis of the data (Table S5) revealed that 101 DEGs were present in both hybrids. Up-regulated DEGs were up-regulated in both, and down-regulated DEGs were down-regulated in both with only one exception. The KEGG pathway enrichment analysis (Figs. 4C and 4D) revealed that the top five regulated pathways contained protein processing in endoplasmic reticulum, which was down-regulated in both Jinfeng X Chenghui 727 and Jinfeng X Chuanhui 907. Further, the pathways of photosynthesis-antenna proteins, photosynthesis, and carbon fixation in photosynthetic organisms were up-regulated in Jinfeng X Chuanhui 907 as observed in Jinfeng B. However, such pathways related to photosynthesis were not observed in Jinfeng X Chenghui 727. But, some of the genes, of course not all, showed also up-regulation (Table S12, S13, and S14) in the hybrid.

The data demonstrate that AtNF-YB1 in rice markedly alters the transcriptome. However, the actual actions may be different depending on the genetic background. The pathway of protein processing in endoplasmic reticulum might be one of the highly impacted pathways. Plant photosynthesis might be significantly affected, as well.

AtNF-YB1 impacts photosynthesis in rice

The discovery of alteration of the photosynthesis pathways further led to measuring photosynthetic traits. Both transgenic Jinfeng B and hybrid T9 showed markedly higher photosynthetic rates. However, transgenic Kasalath and T7 increased or decreased by 5%, respectively (Fig. 5A). These results are consistent with the transcript levels of the genes in the three pathways related to plant photosynthesis from the RNA-seq, indicating the influence of AtNF-YB1 on photosynthesis in rice may depend on the genetic background.

The chlorophyll contents in the leaves were then analyzed. Obtained data showed either chlorophyll a or b was a little increased in the transgenic Kasalath but decreased in the transgenic Jinfeng B (Fig. 5B). However, both hybrids showed a clear increase of both chlorophyll a and b in the transgenic plants (Fig. 5C). The data indicate that enhancement of the photosynthetic pigments in the transgenic hybrids, whereas, in pure line, the effects were quite small, and up-or down-regulation also depends on the genetic background.

AtNF-YB1 promotes flowering under LD conditions via enhancing expression of the florigen, FLOWERING LOCUS T (FT), but not under SD in Arabidopsis (Cai et al. 2007). In this study, the expression of AtNF-YB1 in rice activated the transcriptions of the florigen genes, Hd3a and RFT1 (Komiya et al. 2008), indicating that AtNF-YB1 as a transgene in rice is engaged in the flowering transition. The early flowering of the transgenic rice was observed under both NLD and NSD. Arabidopsis is usually flowering under LD conditions after a cold period of vernalization. However, rice is an SD plant that flowers in summer without vernalization. Therefore, AtNF-YB1 in Arabidopsis might be integrated with other genes after the cold period in response to LD photoperiod to promote flowering (Cai et al. 2007). In rice, AtNF-YB1 increases the expression of both Hd1 and Ehd1, two major upstream integrators of the florigen (Komiya et al. 2009). Ehd1 is truly an activator for flowering transition, but Hd1 works as an activator or inhibitor depending on other transcription factors, such as Ghd7, DTH8, and PRR37 (Nemoto et al. 2016; Zhang et al. 2017; Wang et al. 2019; Fujino et al. 2019).

Ghd7 is a functional allele in Kasalath (Gao et al. 2014) and also functional in all the other tested cultivars (GenBank: OP030525, OP030526, OP030527). DTH8 is a functional allele in Kasalath (Wei et al. 2010b) but defective alleles in 8277B (GenBank: OP030530), Chenghui 727(GenBank: OP030531), and Chuanhui 907(GenBank: OP030532). PRR37 is non-functional alleles in both Kasalath (Murakami et al. 2005) and 82779B (GenBank: OP030533), functional alleles in both Chenghui 727(GenBank: OP030531) and Chuanhui 907(GenBank: OP030532).Therefore, suppressing the expression of the functional Ghd7 is probably the major reason to enhance Ehd1 transcriptions independently on photoperiod so that early flowering happens under both NLD and NSD in either Kasalath or the two hybrids. DTH8 (Wang et al. 2019) and PRR37 (Fujino et al. 2019) have been proved to be suppressors working together with Ghd7 and Hd1, inhibiting Ehd1 transcription so that the expressions of Hd3a and RFT1 are subsequently inhibited. Thus, more studies are needed to examine the early flowering induced by AtNF-YB1 in more cultivars. Notably, the evidence from rice with a combination of the different alleles of Hd1, Ghd7, DTH8, and PRR37 may be required.

The genes in the photoperiod flowering pathways always play pleiotropic effects on plant growth and grain yield (Xue et al. 2008; Wei et al. 2010a; Zhang Z et al. 2019). Early flowering is usually associated with a short growth period, which, in turn, may result in lower yields (Zhu et al. 2017; Cui et al. 2019). Ghd7 markedly influences the plant height, heading date, and grain yield, simultaneously (Lu et al. 2012). In Kasalath Ghd7-Ehd1-Hd3a/RFT may be the only working pathway so that the early flowering finally brings out yield reductions of the transgenic Kasalath. However, more productive panicle is observed in Kasalath and two hybrids, as well.

The gene Hd3a is usually expressed in the phloem tissue of leaves and moves not only to the shoot apical meristem but also to the axillary meristem to promote branching (Tsuji et al. 2015). Thus, highly induced expressions of Hd3a in the transgenic rice plants might lay a foundation for more productive panicles. The grain yield was reduced in the transgenic Kasalath; however, it was similar or even increased in the two transgenic hybrids. The less reduction of the panicle length in the two transgenic hybrids in comparison with Kasalath was clearly observed in both NLD and NSD with lesser in NLD, which might contribute to the less grain reduction and keep the yields for transgenic hybrids. Even though there is a functional Hd1 allele in the hybrids, which is clearly different from the no-functional allele in Kasalath, evidence could not support that keeping grain yield in the hybrids directly resulted from the functional Hd1.

The interactions of Hd1, Ghd7, DTH8, and PRR37 make a complicated scenario, which plays a critical role in the rice growth period and yield (Zhang et al. 2019; Zhang et al. 2019; Sun et al. 2022). However, the longer growth duration for larger use of available temperature and light does not always result in higher grain production. An optimum combination of the flowering time gene alleles must be carefully selected to maximize grain yield in rice (Zhang et al. 2019). The transgenic rice hybrid with the AtNF-YB1 could be a way to get better performance in grain yield with a shorter growth period under NLD. However, under NSD, the grain yields are reduced with the transgene for both hybrids, implying the mechanism that worked in NLD may not work well in NSD. The hybrids, Jinfeng X Chenghui 727 and Jinfeng X Chuanhui 907, are bred cultivars to be used in the field under NLD in Chengdu.

The RNA-seq reveals that expressions of AtNF-YB1in rice impact many biological processes. Despite differences in varieties, the protein processing pathway in endoplasmic reticulum was regulated in all the transgenic rice, with majorities in down-regulation. Notably, all the identified genes from the pathway are significantly down-regulated in the two hybrids. The suppression of the activities related to protein folding, sorting, and degradation in the endoplasmic reticulum suggests alteration of protein metabolism related to plant growth and development in the transgenic plants with the expression of AtNF-YB1. The three pathways of photosynthesis - antenna proteins, and photosynthesis are up-regulated significantly in Jinfeng B and in hybrid Jinfeng X Chuanghui 907. Photosynthesis rate measurement in subsequent experiments confirmed that the expressions of AtNF-YB1 increased photosynthesis in Jingfeng B and in the hybrid Jinfeng X Chuanghui 907. However, the increased chlorophyll does not increase the photosynthetic rate in the hybrid Jinfeng X Chenghui 727. The improved photosynthesis is also quite small in Kasalath. Therefore, the increase in photosynthetic rate might not be directly from the increase in photosynthetic pigments. Several reports demonstrate that lowering chlorophyll contents benefit leaf photosynthesis and improve the light distribution in the canopy resulting in more grain yield (Gu et al. 2017; Slattery et al. 2017; Wang et al. 2022).

AtNF-YB1 in Arabidopsis is a promoter for flowering under LD conditions (Cai et al. 2007), benefits the root growth (Ballif et al. 2011), and is also involved in flowering under osmotic stress (Chen et al. 2007). In maize, the transgenic plants with AtNF-YB1 show drought tolerance and early flowering (Nelson et al. 2007). In this study, AtNF-YB1 expression in rice clearly shows improvement in photosynthesis and grain yield with shorter growth period under NLD, which might be useful for breeding hybrid cultivars with early maturity.

Accession Numbers

These sequence data have been submitted to the NCBI GenBank database under accession numbers from OP030513 to OP030533 and BioProject accession number PRJNA858181.

Acknowledgements

This work was financially supported by Sichuan Provincial Scientific and Technological Funding, No. 2016ZYPZ-002 and No. 2018JY0027.

Author contributions

KC and MP designed experiments and analyzed data. FG, CP, XL, FL, YR, SN, XZ, WT, ZW, XF and MP performed experiments. KC prepared the manuscript. All authors read and approved the manuscript.

Conflict of interest

The authors declare no conflict of interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

An G, Ebert PR, Mitra A, Ha SB (1988) Binary vectors. In: Gelvin SB Schilperoort RA (eds) Plant molecular biology manual vol A3, Kluwer Academic Publishers, Dordrecht, pp 1–19
Ballif J, Endo S, Kotani M, MacAdam J, Wu Y (2011), Over-expression of HAP3b enhances primary root elongation in Arabidopsis. Plant Physiol Biochem 49:579–583
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120
Cai M, Chen S, Wu M, Zheng T, Zhou L, Li C, Zhang H, Wang J, Xu X, Chai J, Ren Y, Guo X, Zhang X, Lei C, Cheng Z, Wang J, Jiang L, Zhai H, Wang H, Zhu S, Wan, J (2019) Early heading 7 interacts with DTH8 and regulates flowering time in rice. Plant Cell Rep 38:521–532
Cai X, Ballif J, Endo S, Davis E, Liang M, Chen D, DeWald D, Kreps J, Zhu T, Wu Y (2007) A putative CCAAT-binding transcription factor is a regulator of flowering timing in Arabidopsis. Plant Physiol 145: 98–105
Chen E, Huang X, Tian Z, Wing RA, Han B (2019) The genomics of Oryza species provides insights into rice domestication and heterosis. Annu Rev Plant Biol 70:639–665
Chen NZ, Zhang XQ, Wei PC, Chen QJ, Ren F, Chen J, Wang XC (2007) AtHAP3b plays a crucial role in the regulation of flowering time in Arabidopsis during osmotic stress. J Biochem Mol Biol 40:1083–1089
Chen S, Songkumarn P, Liu J, Wang GL (2009) A versatile zero background T-vector system for gene cloning and functional genomics. Plant Physiol 150:1111–1121
Cui Y, Li C, Zhou F, Chen H, Zhang W, Ma W, Lin Y (2020) Developing of transgenic glyphosate-tolerant Indica restorer line with commercial application potential. Mol Breeding 40:1–13
Cui Y, Zhu M, Xu Z, Xu Q (2019) Assessment of the effect of ten heading time genes on reproductive transition and yield components in rice using a CRISPR/Cas9 system. Theor Appl Genet 132:1887–1896
Doi K, Izawa T, Fuse T, Yamanouchi U, Kubo T, Shimatani Z, Yano M, Yoshimura A (2004) Ehd1 a B-type response regulator in rice confers short-day promotion of flowering and controls FT-like gene expression independently of Hd1. Genes Dev 18:926–936
Doyle JJ, and Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15
Du A, Tian W, Wei M, Yan W, He H, Zhou D, Huang X, Li S, Ouyang X (2017) The DTH8-Hd1 module mediates day-length-dependent regulation of rice flowering. Mol Plant 10:948–961
Fujino K, Yamanouchi U, Nonoue Y, Obara M, Yano M (2019) Switching genetic effects of the flowering time gene Hd1 in LD conditions by Ghd7 and OsPRR37 in rice. Breed Sci 69:127–132
Gao H, Jin M, Zheng XM, Chen J, Yuan D, Xin Y, Wang M, Huang D, Zhang Z, Zhou K, Sheng P, Ma J, Ma W, Deng H, Jiang L, Liu S, Wang H, Wu C, Yuan L, Wan J (2014) Days to heading 7 a major quantitative locus determining photoperiod sensitivity and regional adaptation in rice. Proc Natl Acad Sci USA 111:16337–16342
Gao H, Zheng X, M, Fei G, Chen J, Jin M, Ren Y, Wu W, Zhou K, Sheng P, Zhou F, Jiang L, Wang J, Zhang X, Guo X, Wang JL, Cheng Z, Wu C, Wang H, Wan JM (2013) Ehd4 encodes a novel and Oryza-genus-specific regulator of photoperiodic flowering in rice. PLoS Genet 9:e1003281
Gómez-Ariza J, ,Galbiati F, Goretti D, Brambilla V, Shrestha R, Pappolla A, Courtois B, Fornara F (2015) Loss of floral repressor function adapts rice to higher latitudes in Europe. J Exp Bot 66:2027–2039
Goretti D, Martignago D, Landini M, Brambilla V, Gómez-Ariza J, Gnesutta N, Galbiati F, Collani S, Takagi H, Terauchi R, Mantovani R, Fornara F (2017) Transcriptional and post-transcriptional mechanisms limit heading date 1 (Hd1) function to adapt rice to high latitudes. PLoS Genet 3:e1006530
Gu J, Zhou Z, Li Z, Chen Y, Wang Z, Zhang H (2017) Rice (Oryza sativa L, ) with reduced chlorophyll content exhibit higher photosynthetic rate and efficiency improved canopy light distribution and greater yields than normally pigmented plants. Field Crop Res 200:58–70
Guo S, Dai S, Singh P, K, Wang H, ,Wang Y, Tan J, L, H, Wee, W, Ito T (2018) A membrane-bound NAC-like transcription factor OsNTL5 represses the flowering in Oryza sativa. Front Plant Sci 9:555
Hu Y, Li S, Xing Y (2019) Lessons from natural variations: artificially induced heading date variations for improvement of regional adaptation in rice. Theor Appl Genet 132:383–394
Kawahara Y, De La Bastide M, Hamilton JP, Kanamori H, McCombie WR, Ouyang S, Schwartz DC, Tanaka T, Wu J, Zhou S, Childs KL, Davidson RM, Lin H, Quesada-Ocampo L, Vaillancourt B, Sakai H, Lee SS, Kim J, Numa H, Itoh T, Buell CR, Matsumoto T (2013) Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice 6:4
Khush GS (1997) Origin dispersal cultivation and variation of rice. Plant Mol Biol 35:25–34
Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat methods 12:357–360
Kim SL, Lee SY, Kim HJ, Nam HG, An GH (2007) OsMADS51 is a short-day flowering promoter that functions upstream of Ehd1 OsMADS14 and Hd3a. Plant Physio, 145:1484–1494
Kim SR, Torollo G, Yoon MR, Kwak J, Lee CK, Prahalada GD, Choi IR, Yeo US, Jeong OY, Jena KK, Lee JS (2018) Loss-of-function alleles of heading date 1 (Hd1) are associated with adaptation of temperate japonica rice plants to the tropical region. Front Plant Sci 9:1827
Kojima S, Takahashi Y, Kobayashi Y, Monna L, Sasaki T, Araki T, Yano M (2002), Hd3a a rice ortholog of the Arabidopsis FT gene promotes transition to flowering downstream of Hd1 under short-day conditions. Plant Cell Physiol 43:1096–1105
Komiya R, Ikegami A, Tamaki S, Yokoi S, Shimamoto K (2008) Hd3a and RFT1 are essential for flowering in rice. Development 135:767–774
Komiya R, Yokoi S, Shimamoto K (2009) A gene network for long day flowering activates RFT1 encoding a mobile flowering signal in rice. Development 136:3443–3450
Koo BH, Yoo SC, Park JW, Kwon CT, Lee BD, An G, Zhang Z, Li J, Li Z, Paek NC (2013) Natural variation in OsPRR37 regulates heading date and contributes to rice cultivation at a wide range of latitudes. Mol Plant 6:1877–1888
Lee S, Kim J, Han JJ, Han M, J, An G (2004) Functional analyses of the flowering time gene OsMADS50 the putative SUPPRESSOR OF OVEREXPRESSION OF CO 1/AGAMOUS-LIKE 20 (SOC1/AGL20) ortholog in rice. Plant J 38:754–764
Li B, Du X, Fei Y, Wang F, Xu Y, Li X, Li W, Chen Z, Fan F, Wang J, Tao Y, Jiang Y, Zhu QH, Yang J (2021) Efficient breeding of early-maturing rice cultivar by editing PHYC via CRISPR/Cas9. Rice 14:86
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550
Lu L, Yan W, Xue W, Shao D, Xing Y (2012) Evolution and association analysis of Ghd7 in rice. PloS one 7:e34021
Matsubara K, Yamanouchi U, Nonoue Y, Sugimoto K, Wang Z, X, Minobe Y, Yano M (2011) Ehd3 encoding a plant homeodomain finger-containing protein is a critical promoter of rice flowering. Plant J 66:603–612
Matsubara K, Yamanouchi U, Wang ZX, Minobe,Y, Izawa T, Yano M (2008) Ehd2 a rice ortholog of the maize INDETERMINATE1 gene promotes flowering by up-regulating Ehd1. Plant Physiol 148:1425–435
Murakami M, Matsushika A, Ashikari M, Yamashino T, Mizuno T (2005) Circadian-associated rice pseudo response regulators (OsPRRs): insight into the control of flowering time. Biosci Biotechnol Biochem 69:410–414
Nelson DE, Repetti PP, Adams TR, Creelman RA, Wu J, Warner DC, Anstrom DC, Bensen RJ, Castiglioni PP, Donnarummo MG, Hinchey BS, Kumimoto RW, Maszle DR, Canales RD, Krolikowski KA, Dotson SB, Gutterson N, Ratcliffe OJ, Heard JE (2007) Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres. Proc Natl Acad Sci USA 104:16450–16455
Nemoto Y, Nonoue Y, Yano M, Izawa T (2016) Hd1 a CONSTANS ortholog in rice functions as an Ehd1 repressor through interaction with monocot-specific CCT-domain protein Ghd7. Plant J 86:221–233
Ogiso-Tanaka E, Matsubara K, Yamamoto SI, Nonoue Y, Wu J, Fujisawa H, Ishikubo H, Tanaka T, Ando T, Matsumoto T, Yano M (2013) Natural variation of the RICE FLOWERING LOCUS T 1 contributes to flowering time divergence in rice. PloS one 8:e75959
Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL (2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat biotechnol 33:290–295
Sahoo KK, Tripathi AK, Pareek A, Sopory SK, Singla-Pareek SL (2011) An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars. Plant methods 7:1–11
Saika H, Toki S (2010) Mature seed-derived callus of the model indica rice variety Kasalath is highly competent in Agrobacterium-mediated transformation. Plant cell rep 29:1351–1364
Slattery RA, VanLoocke A, Bernacchi CJ, Zhu XG, Ort DR (2017) Photosynthesis light use efficiency and yield of reduced-chlorophyll soybean mutants in field conditions. Front Plant Sci 8:549
Sun C, Chen D, Fang J, Wang P, Deng X, Chu C (2014) Understanding the genetic and epigenetic architecture in complex network of rice flowering pathways. Protein cell 5:889–898,
Sun K, Huang M, Zong W, Xiao D, Lei C, Luo Y, Song Y, Li S, Hao Y, Luo W, Xu B, Guo X, Wei G, Chen L, Liu Y, G, Guo J (2022) Hd1 Ghd7 and DTH8 synergistically determine the rice heading date and yield-related agronomic traits. J Genet Genomics 49:437–447
Takahashi Y, Teshima KM, Yokoi S, Innan H, Shimamoto K (2009) Variations in Hd1 proteins Hd3a promoters and Ehd1 expression levels contribute to diversity of flowering time in cultivated rice. Proc Nat Acad Sci USA 106:4555–4560
Tsuji H, Tachibana C, Tamaki S, Taoka K, Kyozuka J, Shimamoto K (2015) Hd3a promotes lateral branching in rice. Plant J 82:256–266
Wang G, Zeng F, Song P, Sun B, Wang Q, Wang J (2022) Effects of reduced chlorophyll content on photosystem functions and photosynthetic electron transport rate in rice leaves. J Plant Physiol 272:153669
Wang P, Gong R, Yang Y, Yu S (2019) Ghd8 controls rice photoperiod sensitivity by forming a complex that interacts with Ghd7. BMC Plant Biol 19:462
Wei FJ, Tsai YC, Wu HP, Huang LT, Chen YC, Chen YF, Wu CC, Tseng YT, Hsing YC (2016) Both Hd1 and Ehd1 are important for artificial selection of flowering time in cultivated rice. Plant Sci242:87–194
Wei X, Liu L, Xu J, Jiang L, Zhang W, Wang J, Zhai H, Wan J (2010a) Breeding strategies for optimum heading date using genotypic information in rice. Mol Breeding 25:287–298
Wei X, Xu J, Guo H, Jiang L, Chen S, Yu C, Zhou Z, Hu P, Zhai H, Wan J (2010b) DTH8 suppresses flowering in rice influencing plant height and yield potential simultaneously. Plant Physiol 153:1747–1758
Wu C, You C, Li C, Long T, Chen G, Byrne ME, Zhang Q (2008) RID1 encoding a Cys2/His2-type zinc finger transcription factor acts as a master switch from vegetative to floral development in rice. Proc Natl Acad Sci USA 105:12915–12920
Wu W, Zheng X, M, Lu G, Zhong Z, Gao H, Chen L, Wu C, Wang HJ, Wang Q, Zhou K, Wang JL, Wu F, Zhang X, Guo X, Cheng Z, Lei C, Lin Q, Jiang L, Wang H, Ge S, Wan J (2013) Association of functional nucleotide polymorphisms at DTH2 with the northward expansion of rice cultivation in Asia. Proc Natl Acad Sci USA 110:2775–80
Xue W, Xing Y, Weng X, Zhao Y, Tang W, Wang L, Zhou H, Yu S, Xu C, Li X, Zhang Q (2008) Natural variation in Ghd7 is an important regulator of heading date and yield potential in rice. Nat Genet 40:761–767
Yan W, Liu H, Zhou X, Li Q, Zhang J, Lu L, Liu T, Liu H, Zhang C, Zhang Z, Shen G, Yao W, Chen H, Yu S, Xie W, Xing Y (2013) Natural variation in Ghd7.1 plays an important role in grain yield and adaptation in rice. Cell Res 23:969–971
Yan,WH, Wang P, Chen HX, Zhou HJ, Li QP, Wang CR, Ding ZH, Zhang YS, Yu SB, Xing YZ, Zhang QF ( 2011) A major QTL Ghd8 plays pleiotropic roles in regulating grain productivity plant height and heading date in rice. Mol Plant 4:319–330
Yano M, Katayose Y, Ashikari M, Yamanouchi U, Monna L, Fuse T, Baba T, Yamamoto K, Umehara Y, Nagamura Y, Sasaki T (2000) Hd1 a major photoperiod sensitivity quantitative trait locus in rice is closely related to the Arabidopsis flowering time gene CONSTANS. Plant Cell 12:2473–2484
Yokoo T, Saito H, Yoshitake Y, Xu Q, Asami T, Tsukiyama T, Teraishi M, Okumoto Y, Tanisaka T (2014) Se14 encoding a JmjC domain-containing protein plays key roles in long-day suppression of rice flowering through the demethylation of H3K4me3 of RFT1. PLoS One 9:e96064
Zhang B, Liu H, Qi F, Zhang Z, Li Q, Han Z, Xing Y (2019) Genetic interactions among Ghd7 Ghd8 OsPRR37 and Hd1 contribute to large variation in heading date in rice. Rice 12:48
Zhang H, Zhu S, Liu T, Wang C, Cheng Z, Zhang X, Chen L, Sheng P, Cai M, Li C, Wang J, Zhang Z, Chai J, Zhou L, Lei C, Guo X, Wang J, Wang J, Jiang L, Wu C, Wan J (2019) DELAYED HEADING DATE1 interacts with OsHAP5C/D delays flowering time and enhances yield in rice, Plant Biotechnol J 17:531–539
Zhang J, Zhou X, Yan W, Zhang Z, Lu L, Han Z, Zhao H, Liu H, Song P, Hu Y (2015) Combinations of the Ghd7 Ghd8 and Hd1 genes largely define the ecogeographical adaptation and yield potential of cultivated rice. New Phytol 208:1056–1066
Zhang ZH, Zhu YJ, Wang SL, Fan YY, Zhuang JY (2019) Importance of the interaction between heading date genes Hd1 and Ghd7 for controlling yield traits in rice. Int J Mol Sci 20:516
Zhang Z, Hu W, Shen G, Liu H, Hu Y, Zhou X, Liu T, Xing Y (2017) Alternative functions of Hd1 in repressing or promoting heading are determined by Ghd7 status under long-day conditions. Sci Rep 7:5388
Zhang Z, Zhang B, Qi F, Wu H, Li Z, Xing Y (2019) Hd1 function conversion in regulating heading is dependent on gene combinations of Ghd7 Ghd8 and Ghd7, 1 under long-day conditions in rice. Mol Breed 39:92
Zhang Z, Zhu Y, Wang S, Fan Y, Zhuang J (2021) Genetic interaction of Hd1 with Ghd7 DTH8 and Hd2 largely determines eco-geographical adaption of rice varieties in southern China. Rice Sci 28:114–118
Zhu YJ, Fan YY, Wang K, Huang DR, Liu WZ, Ying JZ, Zhuang JY (2017), Rice Flowering Locus T 1 plays an important role in heading date influencing yield traits in rice. Sci Rep 7:4918
Zong W, Ren D, Huang M, Sun K, Feng J, Zhao J, Xiao D, Xie W, Liu S, Zhang H, Qiu R, Tang W, Yang R, Chen H, Xie X, Chen L, Liu Y, Guo J (2021) Strong photoperiod sensitivity is controlled by cooperation and competition among Hd1 Ghd7 and DTH8 in rice heading. New Phytol 229:1635–1649
Zhou Y, Xu S, Jiang N, Zhao X, Bai Z, Liu J, Yao W, Tang Q, Xiao G, Lv C, Wang K, Hu X, Tan J, Yang Y (2022) Engineering of rice varieties with enhanced resistances to both blast and bacterial blight diseases via CRISPR/Cas9, Plant Biotechnol J 20:876–885

Table 1 Agronomic traits of the transgenic Kasalath (NLD, Chengdu) †
		Plant height (cm)	Length of main panicle (cm)	Number of productive panicle per plant	Number of filled grain per panicle	1000-grain weight (g)	Weight per plant (g)
First	TK-01	136.2±1.6	21.86±0.1	10.69±1.3	145.15±34.3	17.29±0.6	21.47±3.1
	TK-02	124.3±5.1	21.42±0.6	11.00±3.4	115.24±10.2	14.16±0.5	15.48±5.7
	TK-03	133.4±5.9	21.57±0.7	14.00±0.4	111.00±4.8	16.85±0.8	21.19±1.5
	NT-KA	160.0±4.7	26.71±0.6	10.86±1.2	164.92±6.8	18.51±0.8	27.41±3.4
Second	TK-01	138.0±3.4	24.29±0.4	10.11±0.8	145.03±10.4	17.18±0.3	21.73±3.9
	TK-02	118.2±4.6	22.48±0.5	8.78±1.0	122.97±8.7	15.24±0.4	17.40±1.8
	TK-03	136.6±3.0	23.36±0.3	9.19±0.1	136.50±14.0	16.82±0.3	19.90±5.4
	NT-KA	158.7±1.5	26.56±0.3	9.33±0.1	179.03±3.2	18.03±0.3	23.72±5.3
† TK is trangenic Kasalath. -01, -02 and -03 are three independent transgenic lines. NT-KA is wild-type Kasalath.

Table 2 Agronomic traits of the transgenic hybrids (NLD, Chengdu) †
	Plant height (cm)	Length of main panicle (cm)	Number of productive panicle per plant	Number of filled grain per panicle	Seed setting rate	100-grain weight (g)	Grain weight of 20 plants (Kg)
T7-01	122.69±0.5	28.08±0.2	15.53±1.1	273.00±3.1	90.34±0.9	2.96±0.04	1.08±0.08
T7-02	127.20±0.4	27.91±0.4	14.80±0.8	278.80±2.2	91.70±1.4	2.91±0.04	1.04±0.12
NT727	142.21±1.6	29.57±0.4	11.47±0.5	310.73±28.3	93.85±2.3	3.09±0.06	1.00±0.03
T9-01	123.69±0.8	28.56±0.2	14.07±1.4	319.73±15.6	89.03±0.3	2.97±0.01	0.99±0.10
T9-02	132.69±0.4	28.25±0.8	11.67±0.2	307.00±41.3	97.36±2.5	3.02±0.10	1.18±0.07
NT907	143.09±0.7	28.92±0.6	11.93±0.1	316.00±25.3	86.84±3.1	3.12±0.03	0.92±0.06
† T7 and T9 are the transgenic hybrids, Jinfeng X Chenghui 727 and Jinfeng X Chuanhui 907, respectively. -1 and -02 are two independent transgenic lines. NT727 and NT907 are wild-type hybrids, Jinfeng X Chenghui 727 and Jinfeng X Chuanhui 907, respectively.

Early flowering of the transgenic rice with AtNF-YB1

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