Transcriptome analysis of flowering regulation by sowing date in Japonica Rice (Oryza sativa L.).

Hybrid japonica cultivars, such as the Yongyou series, have shown high yield potential in the field in both the early and late growing seasons. Moreover, understanding the responses of rice flowering dates to temperature and light is critical for improving yield performance. However, few studies have analyzed flowering genes in high-yielding japonica cultivars. Based on the five sowing date experiments from 2019 to 2020, select the sensitive cultivar Yongyou 538 and the insensitive cultivar Ninggeng 4 and take their flag leaves and panicles for transcriptome analysis. The results showed that compared with sowing date 1 (6/16), after the sowing date was postponed (sowing date 5, 7/9), 4480 and 890 differentially expressed genes (DEGs) were detected in the leaves and panicles in Ninggeng 4, 9275 and 2475 DEGs were detected in the leaves and panicles in Yongyou 538, respectively. KEGG pathway analysis showed that both Ninggeng 4 and Yongyou 538 regulated rice flowering through the plant circadian rhythm and plant hormone signal transduction pathways. Gene expression analysis showed that Os01g0566050 (OsELF3-2), Os01g0182600 (OsGI), Os11g0547000 (OsFKF1), Os06g0275000 (Hd1), and Os09g0513500 (FT-1) were expressed higher and Os02g0771100 (COP1-1) was expressed lower in Yongyou 538 compared with Ninggeng 4 as the climate conditions changed, which may be the key genes that regulate the flowering process with the change of temperature and light resources in sensitive cultivar Yongyou 538 in the late season.

Introduction

In recent years, with continuous breakthroughs inpan> pan> class="Chemical">breeding technology and the increase in residents' consumption status, the demand for high-quality rice with high yield and good flavor, especially japonica rice, has been rising[1]. And the area of japonica cultivars on the middle-lower Yangtze Plain has become an increasingly obvious trend in recent decades[2]. However, different rice varieties exhibit regionality, which is determined by the rice heading period. Determining the appropriate heading period is closely related to achieving high and stable rice yields, and it is also the key to determining the appropriate planting area and system[3]. As latitude decreases, the heading stage of rice varieties generally advances, but there are some exceptions, such as Ninggeng 4[4], which is bred in Jiangsu Province. However, its yield is only 6.1–6.8 t ha−1 when planted in Zhejiang Province[5]. Therefore, analyzing the climate adaptability of japonica rice is of great significance for breeding and selecting japonica varieties with wide adaptability and high quality and yield.

n class="Species">Rice flowerinpan>g is a complex biological process that is controlled by the genpan>etic characteristics of the pan> class="Species">rice variety and by the external environment, including the photoperiod[6-8], drought conditions[9-11] and temperature[12-14]. The photoperiod pathway is considered to be the key pathway that regulates rice flowering[15]. Many of the genes involved in the photoperiod pathway in rice flowering have been successfully cloned by QTL mapping methods, such as Heading date 1 (Hd1), Heading date-3a (Hd3a), Early heading date 1 (Ehd1), Rice Indeterminate 1 (RID1), etc. In addition, map-based cloning has revealed that the rice photoperiod pathway is regulated mainly by two independent signal pathways, the OsGI-Hd1-Hd3a pathway[7], which is conserved with Arabidopsis, and unique genes such as Ehd1[16], Ehd2[17], Ehd3[18], Ghd7[19], etc. However, the functions and regulatory pathways of these genes are not fully understood. Previous studies have suggested that Hd3a is highly conserved among rice cultivars, and functional RFT1 exists in most japonica and indica cultivars grown at high latitudes, while nonfunctional RFT1 can be detected in some indica rice varieties grown at lower latitudes[20,21]. In addition, Kim et al.[22] pointed out that the functional alleles of Hd1 were present in most temperate japonica rice varieties, while all 12 tropical-adapted japonica varieties had nonfunctional hd1 alleles, therefore, Hd1 was considered a possible target gene for regulating flowering stage responses in different regions.

The molecular mechanisms of plant flowering have consistently been a global research hotspot. In recent years, scientists have discovered that many genes influence the n class="Species">rice flowerinpan>g stage[23-25]. Moreover, pan> class="Chemical">breeders have developed a large number of high-quality and high-yielding varieties. However, few connections have been made between flowering genes and high-yielding varieties. As the most widely used sequencing technology in genomics research, transcriptome sequencing (RNA-seq) technology can be used to quickly and comprehensively obtain almost all transcript and sequence information for a specific tissue or organ of a species in a specific state. Therefore, in this study, the widely planted cultivars Ninggeng 4 and Yongyou 538 were planted on five sowing dates and two sowing dates were selected as materials for transcriptome analysis. The main objectives of this study were to (1) analyze japonica flowering adaptation in response to climate conditions change and (2) select the key genes regulating flowering adaptability to facilitate the spread of japonica rice to a wider planting area.

Results

Differences in yield and biomass of japonica cultivars among sowing dates

There was significant interaction effect of sowing date and year for the yield (Table 1). With the postponed sowing date, the yield of Ninpan>ggenpan>g 4 remainpan>ed unchanpan>ged or slightly inpan>creased, anpan>d the yield chanpan>ged 9.5% (2019) anpan>d 7.2% (2020) among the five sowinpan>g dates. However, Yongyou 538 showed a trenpan>d of risinpan>g first anpan>d thenpan> decreasinpan>g or decreasinpan>g all the time. This pan> class="Species">indicated that the yield of Yongyou 538 was more sensitive to sowing date, while Ningjing 4 was relatively slow.

Table 1

Difference in biomass and yields of different treatments from 2019 to 2020.

Year	Treatment	Vegetative stage (g m−2)		Reproductive stage (g m−2)		Grain filling stage (g m−2)		Yield (t h m−2)
		Ninggeng 4	Yongyou 538	Ninggeng 4	Yongyou 538	Ninggeng 4	Yongyou 538	Ninggeng 4	Yongyou 538
2019	1	493 ± 48 a	654 ± 52 a	338 ± 23 a	1000 ± 55 a	696 ± 122 b	215 ± 101 b	8.5 ± 0.1 a	9.5 ± 0.7 b
	2	313 ± 8 c	478 ± 26 b	319 ± 31 a	909 ± 73 a	745 ± 44 ab	347 ± 198 b	9.3 ± 0.6 a	10.1 ± 0.5 b
	3	465 ± 36 ab	507 ± 19 b	25 ± 35 c	248 ± 48 c	929 ± 101 a	943 ± 121 a	7.2 ± 0.4 b	9.5 ± 0.4 b
	4	439 ± 28 ab	478 ± 15 b	249 ± 60 ab	463 ± 15 b	654 ± 64 b	915 ± 23 a	8.5 ± 0.5 a	11.6 ± 0.3 a
	5	394 ± 18 bc	395 ± 19 c	217 ± 22 b	516 ± 69 b	790 ± 55 ab	801 ± 35 a	9.1 ± 0.5 a	10.0 ± 0.6 b
2020	1	377 ± 3 ab	553 ± 26 a	452 ± 29 b	564 ± 41 ab	454 ± 91 b	519 ± 54 a	6.7 ± 0.2 ab	9.3 ± 0.4 a
	2	400 ± 14 a	509 ± 35 a	266 ± 39 c	447 ± 84 b	857 ± 88 a	666 ± 155 a	6.1 ± 0.1 b	8.1 ± 0.4 b
	3	351 ± 17 b	428 ± 18 b	482 ± 14 b	629 ± 49 a	359 ± 76 b	596 ± 101 a	6.1 ± 0.3 b	8.7 ± 0.5 ab
	4	343 ± 28 b	398 ± 16 b	331 ± 31 c	577 ± 69 ab	588 ± 118 b	698 ± 86 a	7.2 ± 0.6 a	8.5 ± 0.0 ab
	5	168 ± 9 c	365 ± 16 b	602 ± 26 a	597 ± 39 ab	536 ± 54 b	572 ± 68 a	6.9 ± 0.4 ab	8.0 ± 0.0 b
ANOVA
Year (Y)		107.6***	28.8***	264.0***	9.4**	42.9***	0.8 ns	170.5***	110.4***
Sowing (S)		34.8***	58.2***	26.6***	33.4***	6.0**	18.1***	10.3***	6.5**
Y*S		31.4***	6.0**	63.2***	61.9***	13.3***	13.3***	6.1**	10.6***

Data are mean and sd. Means followed by different letters at the same year are significantly different according to Tukey’s HSD test (P < 0.05).

***Means P < 0.001; **means 0.001 ≤ P < 0.01; *means 0.01 ≤ P < 0.05; ns means P ≥ 0.05.

Data are mean and sd. Means followed by different letters at the n class="Chemical">same year are significanpan>tly differenpan>t accordinpan>g to Tukey’s HSD test (P < 0.05).

There was significant interaction effect of sowing date and year for the biomass during the different stages, too (Table 1). The biomass of Ninpan>ggenpan>g 4 decreased slightly at the vegetative stage, except for the sowinpan>g date 2 inpan> 2019 anpan>d the sowinpan>g date 5 inpan> 2020, anpan>d the coefficienpan>t of variation was 9.1% anpan>d 7.0% inpan> 2019 anpan>d 2020, respectively. While Yongyou 538 showed a decreasinpan>g trenpan>d, anpan>d the coefficienpan>ts of variation was 18.8% anpan>d 17.4% inpan> 2019 anpan>d 2020, respectively. Durinpan>g the reproductive anpan>d grainpan> fillinpan>g period, the biomass of pan> class="Chemical">Ninggeng 4 and Yongyou 538 had no obvious trend among the five sowing dates, and the coefficients of variation was 39.0% and 31.4% at the reproductive stage, 25.2% and 32.2% at the grain filling stage for Ninggeng 4 and Yongyou 538, respectively. It indicated that the biomass was affected by the sowing dates, and the biomass of Yongyou 538 had a significantly higher variation range than that of Ninggeng 4.

Differences in growth stage of japonica cultivars among sowing dates

In general, with the postponed sowing dates, the vegetative stage of Ninpan>ggenpan>g 4 anpan>d Yongyou 538 was shortenpan>ed, while the grainpan> fillinpan>g period was extenpan>ded, anpan>d the reproductive stage was slightly differenpan>t (Table 2). Compared sowinpan>g date 5 with sowinpan>g date 1, the vegetative stage of pan> class="Chemical">Ninggeng 4 was shortened by 3 days and 9 days in 2019 and 2020, respectively, and the effective accumulated temperature was changed by 45 °C and 47 °C in 2019 and 2020, respectively. While Yongyou 538 was shortened by 14 days and 15 days in 2019 and 2020, respectively, and the effective accumulated temperature was reduced by 173 °C and 166 °C in 2019 and 2020, respectively. The results showed that the response of different japonica cultivars to sowing dates was significantly different, and the difference in the vegetative stage of Ninggeng 4 was significantly lower than that of Yongyou 538, which indicated that Ninggeng 4 was insensitive to temperature and light, while Yongyou 538 was sensitive.

Table 2

Difference in growth period (effective accumulated temperature) of different treatments from 2019 to 2020.

Year	Treatment	Vegetative stage d (°C)		Reproductive stage d (°C)		Grain filling stage d (°C)		Growth period d (°C)
		Ninggeng 4	Yongyou 538	Ninggeng 4	Yongyou 538	Ninggeng 4	Yongyou 538	Ninggeng 4	Yongyou 538
2019	1	51 (890)	64 (1150)	20 (395)	24 (408)	35 (556)	49 (594)	106 (1841)	137 (2153)
	2	49 (883)	59 (1080)	19 (374)	25 (427)	43 (641)	52 (575)	111 (1899)	136 (2082)
	3	49 (918)	53 (998)	21 (367)	26 (446)	39 (581)	54 (557)	109 (1866)	133 (2001)
	4	48 (911)	55 (1046)	20 (338)	20 (329)	40 (563)	58 (557)	108 (1811)	133 (1932)
	5	48 (935)	50 (977)	17 (273)	23 (365)	43 (557)	59 (514)	108 (1765)	132 (1856)
2020	1	51 (850)	61 (1059)	24 (522)	27 (555)	43 (641)	53 (597)	118 (2014)	141 (2211)
	2	49 (826)	59 (1039)	23 (498)	25 (498)	45 (622)	54 (577)	117 (1946)	138 (2114)
	3	47 (826)	54 (983)	22 (464)	25 (487)	48 (613)	55 (558)	117 (1903)	134 (2028)
	4	45 (821)	50 (937)	22 (219)	25 (461)	49 (563)	59 (564)	116 (1829)	134 (1962)
	5	42 (803)	46 (893)	24 (454)	29 (472)	51 (528)	56 (512)	117 (1786)	131 (1876)

Correlation analysis

Table 3 showed the correlation analysis between the yield and the temperature and light resources (effective accumulated temperature, accumulated ligth hours and solar radiation) of Ninpan>ggenpan>g 4 anpan>d Yongyou 538 durinpan>g the vegetative stage from 2019 to 2020. The results showed that the yield of pan> class="Chemical">Ninggeng 4 was significantly positively correlated with the effective accumulated temperature and light hours at this stage, and the correlation coefficients were 0.7 and 0.8, respectively. While Yongyou 538 was significantly positively correlated with the accumulated light hours at this stage, and the correlation coefficient was 0.7. This indicated that the stable yield of Ninggeng 4 was due to its insensitivity to the vegetative stage, while the yield of Yongyou 538 decreased with the shorted vegetative period.

Table 3

Relation between yield and temperature and light resources in vegetative stage of different treatments from 2019 to 2020.

Variety	Effective accumulated temperature	Accumulated ligth hours	Accumulated solar radiation
Ninggeng 4	0.7*	0.8**	0.6 ns
Yongyou 538	0.5 ns	0.7*	0.4 ns

Sequencing quality analysis of japonica rice tissue

The clean reads from the leaves and panicles of the n class="Species">japonica cultivars pan> class="Chemical">Ninggeng 4 and Yongyou 538 sown on the two sowing dates (CK vs. T) are presented in Table 4 and Table S1. In the leaves, the number of clean reads from Ninggeng 4 in the T treatment was the highest and was 0.43%, 12.85% and 16.37% higher than those from Ninggeng 4 in the CK treatment and Yongyou 538 in the CK and T treatment, respectively. In addition, in the panicles, the number of clean reads from Ninggeng 4 in the T treatment was the highest and was 4.76%, 8.68% and 2.11% higher than those from Ninggeng 4 and Yongyou 538 in the CK treatment and Yongyou 538 in the T treatment, respectively. The number of bases with a mass greater than 20 or 30 in the leaves and panicles accounted for more than 95% of the total number of bases, and the clean reads mapped to the reference genome (Nipponbare) were above 94% as well, indicating that the quality of RNA-seq was sufficient for the subsequent analysis.

Table 4

Comparison of the sequencing data for the two japonica cultivars sown on two sowing dates with the reference genome.

Sample	Cultivar	Treatment	Clean reads	Q20 (%)	Q30 (%)	Total mapped
Leaf	Ninggeng 4	CK	52,871,935	98.92	96.34	51,199,106 (96.84%)
		T	53,099,148	98.95	96.44	51,340,507 (96.69%)
	Yongyou 538	CK	47,050,997	98.95	96.49	45,200,802 (96.07%)
		T	45,630,931	98.50	95.36	43,236,611 (94.75%)
Panicle	Ninggeng 4	CK	49,277,341	98.84	96.14	46,713,541 (94.80%)
		T	51,622,847	98.89	96.27	49,835,160 (96.54%)
	Yongyou 538	CK	47,498,167	98.91	96.35	44,736,859 (94.19%)
		T	50,557,107	98.92	96.39	48,051,996 (95.04%)

Total reads: The number of sequences after filtering (clean reads); Q20, Q30: The percentage of bases with quality greater than 20 or 30; Total mapped: Number of clean reads that can be mapped to the genome.

Comparison of the sequencing data for the two n class="Species">japonica cultivars sownpan> onpan> two sowinpan>g dates with the referenpan>ce genpan>ome.

Total reads: The number of sequences after filtering (clean reads); Q20, Q30: The percentage of bases with quality greater than 20 or 30; Total mapped: n class="Chemical">Number of cleanpan> reads that canpan> be mapped to the genpan>ome.

Differentially expressed genes (DEGs) under the two sowing dates

The two treatments in the leaves and panicles were compared to identify the DEGs between cultivars [Ninpan>ggenpan>g 4, CK (pan> class="Chemical">NCK) vs. Yongyou 538, CK (YCK); Ninggeng 4, T (NT) vs. Yongyou 538, T (YT)] and sowing dates (NCK vs. NT, YCK vs. YT) (Fig. 1A,B). Comparing the NCK and YCK libraries revealed 4747 and 814 DEGs in the leaves and panicles, respectively, including 53.9% upregulated and 46.1% downregulated genes in the leaves and 48.9% upregulated and 51.1% downregulated genes in the panicles. Comparing NT and YT revealed 9622 and 2775 DEGs in the leaves and panicles, respectively, including 49.2% upregulated and 50.8% downregulated genes in the leaves and 48.8% upregulated and 51.2% downregulated genes in the panicles. Comparing NCK with NT revealed 4480 and 890 DEGs in the leaves and panicles, respectively, of which 64.2% and 12.8% were upregulated genes and 35.8% and 87.2% were downregulated genes, respectively. Comparing YCK with YT revealed 9275 and 2475 DEGs in the leaves and panicles, respectively, of which 52.3% and 32.8% were upregulated genes and 47.7% and 67.2% were downregulated genes, respectively.

Figure 1

DEGs in the leaves (A) and panicles (B); flowering DEGs in the leaves (C) and panicles (D); and Venn diagrams of the DEGs in the leaves (E) and panicles (F) among the pairwise comparisons. This figure was created using Microsoft Excel 2010 and Adobe Illustraor CS6.

The results of a functional query with the keyword “flowering” for the DEGs showed that when the sowing date was delayed 23 days, there were 7 upregulated and 8 downregulated flowering DEGs in the leaves and 4 downregulated flowering DEGs in the panicles of Ninpan>ggenpan>g 4. In Yongyou 538, there were 16 upregulated anpan>d 22 downregulated flowerinpan>g DEGs inpan> the leaves anpan>d 3 upregulated anpan>d 8 downregulated flowerinpan>g genpan>es inpan> the panpan>icles (Fig. 1C,D). The Venpan>n diagram of the flowerinpan>g DEGs pan> class="Species">indicated that 6 and 1 flowering DEGs were found in the leaves and panicles of the two japonica cultivars, respectively; 9 and 3 flowering DEGs were found only in the leaves and panicles of Ninggeng 4, respectively; and 32 and 10 flowering DEGs were found only in the leaves and panicles of Yongyou 538, respectively (Fig. 1E,F).

Analysis of flowering DEGs by KEGG pathway

KEGG pathway analysis was carried out to further analyze the function of flowering DEGs in the leaves and panicles of n class="Chemical">Ninpan>ggenpan>g 4 anpan>d Yongyou 538 sown on the two sowinpan>g dates (Fig. 2). In pan> class="Chemical">Ninggeng 4, a total of 6 flowering DEGs (2 upregulated genes Os11g0547000 and Os06g0552900, 4 downregulated genes Os09g0513500, Os06g0157500, Os03g0856700 and Os10g0463400) in the leaves from NCK vs NT were assigned to 3 KEGG pathways, including the plant circadian rhythm, diterpenoid biosynthesis and plant hormone signal transduction pathways, but no pathway was detected for the flowering DEGs in the panicles. On the other hand, in Yongyou 538, a total of 22 flowering DEGs (13 upregulated and 9 downregulated) in the leaves under YCK vs YT were assigned to 14 KEGG pathways, while only 2 upregulated genes (Os01g0182600 and Os11g0547000)in the panicles were assigned to the plant circadian rhythm pathway. Compared with the plant circadian rhythm pathway mapped in the panicles, the upregulated genes in the leaves were significantly enriched in the plant circadian rhythm, glycerolipid metabolism, glycosphingolipid biosynthesis-globin and isoglobin series pathways, while the downregulated genes were involved in porphyrin and chlorophyll metabolism, brassinosteroid biosynthesis, glutathione metabolism, cysteine ​​and methionine metabolism, plant hormone signal transduction, plant circadian rhythm and ubiquitin mediated proteolysis pathways; of these, the porphyrin and chlorophyll metabolism pathways were significantly enriched.

Figure 2

KEGG pathways of the flowering DEGs in the leaves and panicles of Ninggeng 4 and Yongyou 538 in response to different sowing dates. This figure was created using Origin 2021.

KEGG pathways of the flowering DEGs in the leaves and panicles of n class="Chemical">Ninpan>ggenpan>g 4 anpan>d Yonpan>gyou 538 inpan> responpan>se to differenpan>t sowinpan>g dates. This figure was created usinpan>g Originpan> 2021.

DEGs in the plant circadian rhythm pathway

The plant circadian rhythm pathway and the DEGs under the CK vs T in the leaves and panicles of Ninpan>ggenpan>g 4 anpan>d Yongyou 538 are listed inpan> Figs. 3 anpan>d 4 anpan>d Table S2. The results showed that a total of 12 DEGs were inpan>volved. In the leaves, CRY anpan>d pan> class="Gene">COP1 were significantly downregulated, while FKF1 and FT were significantly upregulated in both Ninggeng 4 and Yongyou 538, and the DEGs Os09g0513500 (FT-3) and Os06g0157500 (RFT1) were both downregulated in Ninggeng 4. In addition, Os01g0566050 (OsELF3-2), Os01g0182600 (OsGI), and Os06g0275000 (Hd1) were significantly upregulated in Yongyou 538. In the panicles, only the downregulated DEGs Os02g0114900 (COP1-2) and upregulated DEGs Os01g0182600 (OsGI) and Os11g0547000 (OsFKF1) were found in Yongyou 538.

Figure 3

Schematic diagram of part of plant circadian rhythm pathway (map04712)[26]. This figure was created using Microsoft PowerPoint 2010.

Figure 4

DEGs in the plant circadian rhythm pathway in the leaves (A) and panicles (B) of Ninggeng 4 and Yongyou 538 sown on two sowing dates. This figure was created using Origin 2021.

DEGs in the plant circadian rhythm pathway in the leaves (A) and panicles (B) of n class="Chemical">Ninpan>ggenpan>g 4 anpan>d Yonpan>gyou 538 sownpan> onpan> two sowinpan>g dates. This figure was created usinpan>g Originpan> 2021.

DEGs in the plant hormone signal transduction pathway

Among the pathways associated with the flowering DEGs under the CK vs T, the plant hormone signal transduction pathway was enriched in both Ninpan>ggenpan>g 4 anpan>d Yongyou 538. As shown inpan> Table 5, there were 61 anpan>d 97 DEGs inpan> the leaves inpan> pan> class="Chemical">Ninggeng 4 and Yongyou 538, respectively, and the IAA pathway had the most DEGs in both cultivars, accounting for 24.6% and 24.7% of the total number of DEGs, respectively, in which the reported genes Os01g0741900 (OsIAA6) and Os01g0764800 (OsGH3-2) were differentially expressed both in Ninggeng 4 and Yongyou 538. On the other hand, we found that two flowering DEGs were significantly downregulated in the CTK pathway, including Os10g0463400 (B-ARR) in Ninggeng 4 and Os10g0362300 (CRE1) in Yongyou 538. In addition, Os01g0718300 (OsBRI1), which is involved in the BR pathway, was significantly downregulated in Yongyou 538. Although there were 19 and 35 DEGs in the panicles of Ninggeng 4 and Yongyou 538, respectively, no flowering DEGs were found in the panicles.

Table 5

DEGs in the plant hormone signal transduction pathway in the leaves and panicles of Ninggeng 4 and Yongyou 538.

Plant hormone	NCK versus NT				YCK versus YT
	Leaf		Panicle		Leaf		Panicle
	Upregulated	Downregulated	Upregulated	Downregulated	Upregulated	Downregulated	Upregulated	Downregulated
IAA	4	11	0	1	12	12	1	5
CTK	1	6	0	3	2	9	1	2
GA	0	4	0	1	1	5	0	1
ABA	6	2	0	1	13	7	3	7
ETH	2	7	0	2	4	9	1	2
BR	1	2	0	0	3	4	1	0
JA	7	1	0	9	0	11	0	8
SA	4	3	0	2	3	2	1	2

IAA: Auxin; CTK: Cytokinin; GA: Gibberellin; ABA: Abscisic acid; ETH: Ethylene; BR: Brassinosteroid; JA: Jasmonic acid; SA: Salicylic acid.

DEGs in the plant hormone signal transduction pathway in the leaves and panicles of n class="Chemical">Ninpan>ggenpan>g 4 anpan>d Yonpan>gyou 538.

n class="Chemical">IAA: Auxinpan>; CTK: Cytokinpan>inpan>; pan> class="Chemical">GA: Gibberellin; ABA: Abscisic acid; ETH: Ethylene; BR: Brassinosteroid; JA: Jasmonic acid; SA: Salicylic acid.

qRT-PCR for DEGs

Four DEGs involved in the plant circadian rhythm pathway were analyzed by qRT-PCR. The results showed that the expression patterns detected in the qRT-PCR were similar to those detected with RNA-seq (Fig. 5), pan> class="Species">indicating that the RNA-seq data used in this experiment were reliable.

Figure 5

The relative expression of DEGs in the leaves (A–D) and panicles (E,F) of Ninggeng 4 and Yongyou 538 sown on two sowing dates. This figure was created using Origin 2021 and Adobe Illustraor CS6. Values are the means and sds. Means followed by different letters in the same test method are significantly different according to Tukey’s HSD test (P < 0.05).

The relative expression of DEGs in the leaves (A–D) and panicles (E,F) of n class="Chemical">Ninpan>ggenpan>g 4 anpan>d Yongyou 538 sown on two sowinpan>g dates. This figure was created usinpan>g Originpan> 2021 anpan>d Adobe Illustraor CS6. Values are the meanpan>s anpan>d pan> class="Chemical">sds. Means followed by different letters in the same test method are significantly different according to Tukey’s HSD test (P < 0.05).

Discussion

In our study, when the sowing date was delayed by 23 days, the days-to-heading of Ninpan>ggenpan>g 4 anpan>d Yongyou 538 were occurred 6 anpan>d 15 days earlier thanpan> that inpan> the sowinpan>g date 1 (CK), respectively. In the differenpan>t growth stages, the vegetative anpan>d reproductive periods of pan> class="Chemical">Ninggeng 4 were both decreased by 3 days, while the vegetative and reproductive stages of Yongyou 538 were decreased by 14 and 1 day, respectively (Table 2). This means that the change in the days-to-heading occurred mainly in the vegetative growth period, which is consistent with the results of a previous study[27,28]. During the vegetative stage, the average daily temperatures for Ninggeng 4 and Yongyou 538 under the T treatment were 29.5 °C, which was 7.4% and 5.6% higher than those in the CK treatment and close to the optimum temperature for japonica growth and development (30 °C) (Fig. 6). Duan et al.[29] pointed out that the growth rate of japonica was accelerated and its growth stage was shortened when the average daily temperature increased during the vegetative growth period and that japonica showed different degrees of temperature sensitivity. Similarly, the sensitivity of the responses of Ninggeng 4 and Yongyou 538 to temperature and light changes were not consistent, and Yongyou 538 was more sensitive than Ninggeng 4. The greater number of DEGs in Yongyou 538 than in Ninggeng 4 also supports this idea (Fig. 1A–D). In addition, more DEGs were detected in the leaves than in the panicles (Fig. 1A–D), indicating that leaves, rather than panicles, are the organs that perceive climate conditions change. This finding is similar to that in a previous study showing that plants perceive the change in light hours with their leaves and initiate stem apex growth and flower formation based on these perceptions[30].

Figure 6

Changes in the climate resources from 2019 to 2020. This figure was created using Microsoft Excel 2010.

The biological clock of plants plays an important role in regulating their flowering process. Under different photoperiod treatments, the plant flowering stages can be affected by the genes involved in the plant circadian rhythm pathway. In our study, as the climate conditions changed, DEGs under CK versus T in the circadian rhythm pathways were observed in both Ninpan>ggenpan>g 4 anpan>d Yongyou 538 (Fig. 2). The meteorological data durinpan>g this period showed that the average daily light hours decreased by 25.4% anpan>d 6.3% anpan>d the accumulated light hours decreased by 36.6% anpan>d 10.2% from panpan>icle inpan>itiation to headinpan>g inpan> pan> class="Chemical">Ninggeng 4 and Yongyou 538, respectively (Fig. 6). A previous study showed that when the blue light receptor CRY in plants receives blue light signals, the activity of its downstream gene COP1 is inhibited[31]. As a result, COP1 was significantly downregulated both in Ninggeng 4 and Yongyou 538 in the sowing date 5 (T) compared with sowing date 1 (CK) (Fig. 4). COP1 can interact with its downstream gene ELF3 and regulate the biological activity of light signals input by the downstream target GI to regulate the circadian rhythm pathway and the flowering stage[32]. In addition, OsELF3-2 was significantly upregulated in Yongyou 538 (Fig. 3). Yu et al.[32] pointed out that the function of ELF3 in regulating plant circadian rhythms required complete COP1 activity. Therefore, the downregulation of COP1 in Yongyou 538 resulted in increased expression of GI (Fig. 4). As a gate switch for light signal inputs, the homologous gene of GI in Oryza sativa, OsGI, activates the transcription of Hd1 (the homologous gene of CO in Oryza sativa) under short-day light conditions[7]. In addition, GI can combine with FKF1 to form a blue-light-dependent protein complex, which can indirectly promote the transcription of CO by degrading CDFs (the transcription repressors for CO)[33-35]. Subsequently, CO can promote the differentiation of flower primordia to form flower tissues by inducing the transcription of FT[36]. Therefore, OsFKF1, Hd1 and FT-1 were upregulated with the upregulation of the upstream gene GI in Yongyou 538 in our study (Fig. 3). In Ninggeng 4, OsFKF1 and FT, including FT-2, FT-3 and RFT1, expressed differentially compared CK and T, while OsGI was not differentially expressed (Fig. 4), resulting in a small change in the number of protein complexes formed by FKF1 and GI. As a result, the interaction of upregulated genes and downregulated genes in FT may be the main reason for the difference in the flowering stage of Ninggeng 4 under the two sowing dates.

In addition to their biological clocks, plants can also regulate the flowering process with hormones. Our research found that B-ARR was significantly downregulated in Ninpan>ggenpan>g 4 through the CTK tranpan>sduction pathway. Previous studies have shown that cytokinpan>inpan>s canpan> regulate the pan> class="Species">rice flowering process through genes such as AHK2 and AHK3[37], B-ARR and A-ARR[38]. In Oryza sativa, Ehd1 is highly homologous to B-ARR and can interact with the A-ARR protein OsRR1 to form a heterodimer and inhibit the activity of Ehd1[38]. In terms of photoperiod regulation, Ehd1 can directly regulate its downstream gene FT and promote the flowering stage under short-day light conditions[16]. Therefore, two downregulated genes in FT were detected in Ninggeng 4 (Fig. 4). In addition, BRI1 was significantly downregulated in Yongyou 538 through the BR transduction pathway. In this pathway, BRI1 is a receptor gene on the membrane and can promote dark morphogenesis and early flowering[39]. However, the effect of BRI1 in Yongyou 538 may have been weakened by other flowering genes in our study. In addition, DEGs also existed in the signal transduction pathways of IAA, GA, ABA, Eth, JA, and SA (Table 5), which have been confirmed to play a role in rice flowering[40-44]. The number of hormone-related DEGs, especially those related to IAA, was higher in the sensitive cultivar Yongyou 538 than in the insensitive cultivar Ninggeng 4. This result indicates that hormones play an important role in japonica flowering in response to climate conditions change.

Two different subspecies of n class="Species">rice were selected as the materials for this research. A previous study showed that the functional diversity of flowerinpan>g-time genpan>e sequenpan>ces has no special relationship betweenpan> pan> class="Species">rice subspecies[45]. Gene expression analysis showed that the expression of Os01g0566050 (OsELF3-2), Os11g0547000 (OsFKF1), Os01g0182600 (OsGI), Os06g0275000 (Hd1) and Os09g0513500 (FT-1) was slightly or significantly higher in the sensitive cultivar Yongyou 538 after the sowing date delay (YT) than other treatments (NCK, NT and YCK), while the expression of Os02g0771100 (COP1-1) was significantly lower, these may be the key genes that regulate the flowering process of japonica cultivars adapting to different planting areas. However, this finding needs to be verified by further experiments.

Conclusions

The flowering stage in the two n class="Species">japonica varieties responds differenpan>tly to climate chanpan>ge. Yongyou 538 is more senpan>sitive anpan>d affected by the delay inpan> the sowinpan>g date thanpan> pan> class="Chemical">Ninggeng 4, as indicated by its higher number of DEGs. Therefore, Yongyou 538 can adapt to climate conditions change by modulating the plant circadian rhythm and hormone signal transduction pathways. Gene expression analysis indicated that Os01g0566050 (OsELF3-2), Os01g0182600 (OsGI), Os11g0547000 (OsFKF1), Os06g0275000 (Hd1), and Os09g0513500 (FT-1) were expressed higher and Os02g0771100 (COP1-1) was expressed lower in the sensitive cultivar as the climate conditions changed, these may be the key genes that regulate the flowering process in japonica cultivars adapting to different planting areas.

Materials and methods

Study site description

Field experiments were carried out in 2019 and 2020 at the farm (30°05′n class="Chemical">N, 119°90′E, 21 m altitude) of the Chinpan>a pan> class="Chemical">National Rice Research Institute in Hangzhou, Zhejiang Province. The field soil properties were as follows: organic matter, 30.5 g kg−1, tatal N, 1.7 g kg−1, available P, 14.2 mg kg−1, available K, 140.2 mg kg−1, and pH, 5.8. The weather information, including the average temperature, rainfall, light hours and solar radiation from May to November of 2019 and 2020, are shown in Fig. 6.

Experiment design

The n class="Species">rice varieties pan> class="Chemical">Ninggeng 4 (N) and Yongyou 538 (Y) were selected as the experimental materials, and five sowing dates were conducted in 2019 and 2020. Pregerminated seeds were sown in seedbeds. The sowing and transplanting date were listed in Table 6. The hill spacing was 25 cm × 16.7 cm. The total N application rate was 202.5 kg ha−1, 50% was applied 1 day before transplanting (N:P2O5:K2O 15:15:15; compound fertilizer), 30% was applied 14 days after transplanting (urea), and 20% was applied at the panicle initiation stage (urea). Approximately 100.0 kg ha−1 P2O5 and K2O in the form of compound fertilizer were applied as basal fertilizer 1 day before transplanting, and an additional 65.0 kg ha−1 K2O in the form of potassium chloride was applied as a topdressing at the panicle initiation stage.

Table 6

Sowing and transplanting date of different treatments from 2019 to 2020.

Year	Treatment	Sowing date	Transplanting date
2019	1	2019/6/16	2019/7/3
	2	2019/6/21	2019/7/9
	3	2019/6/27	2019/7/17
	4	2019/7/2	2019/7/23
	5	2019/7/9	2019/7/30
2020	1	2020/6/10	2020/6/29
	2	2020/6/17	2020/7/6
	3	2020/6/24	2020/7/13
	4	2020/7/1	2020/7/20
	5	2020/7/8	2020/7/27

Measuerments

In 2019 and 2020, 6 hills were n class="Chemical">sampled from each plot at the tillerinpan>g (approximately 15–20 days after tranpan>splanpan>tinpan>g), panpan>icle inpan>itiation, headinpan>g, anpan>d maturity stage. The pan> class="Chemical">samples were separated into leaf, stem and panicles and then oven dried for 72 h at 75 °C. The aboveground biomass was calculated by summing the dry weight of all organs. The yield was converted to 14% moisture content. The heading stage was defined as the date when 80% of the panicles had emerged, and the plant reached maturity when 95% of the spikelets had turned yellow.

The flag leaves and panicles of n class="Chemical">Ninpan>ggenpan>g 4 anpan>d Yonpan>gyou 538 inpan> sowinpan>g date 1 (conpan>sidered a conpan>trol check, CK) anpan>d sowinpan>g date 5 (conpan>sidered a treatmenpan>t, T) inpan> 2019, respectively, were removed at the flowerinpan>g stage, packed inpan> tinpan>foil, quickly placed inpan> liquid pan> class="Chemical">nitrogen, and then stored in a − 80 °C freezer for subsequent RNA extraction. Three biological replicates of each sample were taken.

cDNA library preparation and RNA-seq

The lin class="Chemical">brary was constructed usinpan>g anpan> Illuminpan>a TruSeq TM Rpan> class="Chemical">NA Sample Prep Kit method. Total RNA was extracted from the leaves and panicles of Ninggeng 4 and Yongyou 538 from the CK and T treatments. Its concentration and purity were detected by a Nanodrop2000. The integrity of the RNA was detected by agarose gel electrophoresis, and the RNA integrity number (RIN) value was detected by Agilent 2100. Then mRNA was enriched by oligo-dT selection, and fragmentation buffer was added to fragment the mRNA. Under the action of reverse transcriptase, a random six-base primer was added, and cDNA was synthesized by reverse transcription using mRNA as a template. The cDNA was then connected to the adaptor. The library was enriched and amplified in 15 PCR cycles. The target band recovered from the 2% agarose gel was quantified by TBS380 Picogreen and mixed in the indicated ratios. Clusters were generated by bridge PCR amplification on cBot. Finally, the PE library was sequenced on the Illumina platform with a read length of 2 × 150 bp.

Information analysis

Tables and figures were processed by Microsoft Excel 2010 and Microsoft PowerPoint 2010 ((Microsoft, Redmond, WA, Un class="Chemical">SA), Originpan> 2021 (Originpan>Lab, pan> class="Chemical">Northampton, MA, USA) or Adobe Illustraor CS6 (Adobe, San Jose, CA, USA). SAS 9.4 (SAS Institute Inc., Cary, NC, USA) was used to analysis of variance (ANOVA). Two-way ANOVA was used to compare the effects of year and sowing date with a general linear model (GLM). One-way ANOVA was performed to evaluate the effects of sowing date on biomass, yield and growth period parameters. Means were subjected to Tukey’s honestly significant difference (HSD) test at the 0.05 probability level. The raw reads obtained by Illumina sequencing were filtered to obtain high-quality sequencing data (clean reads). TopHat2 was used to compare the clean data with the japonica reference genome Nipponbare (https://www.ncbi.nlm.nih.gov/assembly/GCF_001433935.1/) for gene sequence comparison, obtain the mapped data and evaluate the quality of the sequence results. The gene expression amounts were calculated and normalized as transcripts per million (TPM). The thresholds of |log2FC|> = 1 and padjust < 0.05 were used to screen for differentially expressed genes (DEGs) with DESeq2. Then, the DEGs were associated with KEGG pathways, which were considered significantly enriched at P value_uncorrected < 0.05. The sequencing and preliminary analysis in this experiment were completed by Shanghai Majorbio Biopharm Technology Co., Ltd.

Real-time fluorescence quantitative PCR (qRT-PCR) analysis

Four DEGs involved in KEGG pathways were further analyzed by qRT-PCR. A ChamQ SYn class="Chemical">BR Color Master Mix (2X) (pan> class="Chemical">Nanjing Novazan Biotechnology Co., Ltd.) reverse transcription kit was used in the ABI 7500 Real-Time PCR System. The internal reference gene was actin, and the relative expression of the genes was calculated by the 2−∆ ∆Ct method. The primer sequences are shown in Table 7.

Table 7

qRT-PCR primers.

Gene	Forward primer	Reverse primer
Os11g0547000	5′-GATGAGCACCAAGATGTTAGG-3′	5′-CAGGCTTTGCAGATTCCAG-3′
Os01g0566050	5′-AACAGCGAGCATCAAGCG-3′	5′-CACCAATGGCACCAACAA-3′
Os01g0182600	5′-CAGCGGAAGATTACGATT-3′	5′-CGATGATACATAGCCACCT-3′
Os02g0771100	5′-ACTTTGTTGGGCTGTCTG-3′	5′-TAGGGCTATCGCTCTTCC-3′

Ethics declarations

Experimental research and field studies on cultivars have complied with relevant institutional, national, and international guidelines and legislation. And all cultivars were purchased through formal channels.

Supplementary Table S1.

Supplementary Table S2.