Heading Date In Rice Research Articles

A 9.5-kb deletion in the 1st intron of OsMADS51 enhances temperature sensitivity in rice

Temperature is an important environmental factor affecting heading date of rice. Despite its importance, genes responsible for temperature-sensitive heading in rice have remained elusive. Our previous study identified a quantitative trait locus qHd1 which advances heading date under high temperatures. A 9.5-kb insertion was found in the first intron of OsMADS51 in indica variety Zhenshan 97 (ZS97). However, the function of this natural variant in controlling temperature sensitivity has not been verified. In this study, we used CRISPR/Cas9 to knock out the 9.5-kb insertion in ZS97. Experiments conducted under cotrolled conditions in phytotrons confirmed that deletion increased temperature sensitivity and advanced heading by downregulating the expression level of OsMADS51. One-hybrid assays in yeast, ChIP-quantitative polymerase chain reaction, electrophoretic mobility shift, and luciferase-based transient transactivation assays collectively confirmed that OsMADS51 affects heading date by regulation of heading date gene Ehd1. We further determined that the long non-coding RNA HEATINR is generated from the first intron of OsMADS51, offering an explanation for how the 9.5-kb insertion affects temperature sensitivity. We also found that OsMADS51 was strongly selected in early/late-season rice varieties in South China, possibly accounting for their strong temperature sensitivity. These insights not only advance our understanding of the molecular mechanisms underlying the temperature-responsive regulation of heading date in rice but also provide a valuable genetic target for molecular breeding.

Phosphatidyl Ethanolamine Binding Protein FLOWERING LOCUS T-like 12 (OsFTL12) Regulates the Rice Heading Date under Different Day-Length Conditions.

Plant FLOWERING LOCUS T-Like (FTL) genes often redundantly duplicate on chromosomes and functionally diverge to modulate reproductive traits. Rice harbors thirteen FTL genes, the functions of which are still not clear, except for the Hd3a and RFT genes. Here, we identified the molecular detail of OsFTL12 in rice reproductive stage. OsFTL12 encoding protein contained PEBP domain and localized into the nucleus, which transcripts specifically expressed in the shoot and leaf blade with high abundance. Further GUS-staining results show the OsFTL12 promoter activity highly expressed in the leaf and stem. OsFTL12 knock-out concurrently exhibited early flowering phenotype under the short- and long-day conditions as compared with wild-type and over-expression plants, which independently regulates flowering without an involved Hd1/Hd3a and Ehd1/RFT pathway. Further, an AT-hook protein OsATH1 was identified to act as upstream regulator of OsFTL12, as the knock-out OsATH1 elevated the OsFTL12 expression by modifying Histone H3 acetylation abundance. According to the dissection of OsFTL12 molecular functions, our study expanded the roles intellectual function of OsFTL12 in the mediating of a rice heading date.

Improving rice eating and cooking quality by enhancing endogenous expression of a nitrogen-dependent floral regulator.

Improving rice eating and cooking quality (ECQ) is one of the primary tasks in rice production to meet the rising demands of consumers. However, improving grain ECQ without compromising yield faces a great challenge under varied nitrogen (N) supplies. Here, we report the approach to upgrade rice ECQ by native promoter-controlled high expression of a key N-dependent floral and circadian clock regulator Nhd1. The amplification of endogenous Nhd1 abundance alters rice heading date but does not affect the entire length of growth duration, N use efficiency and grain yield under both low and sufficient N conditions. Enhanced expression of Nhd1 reduces amylose content, pasting temperature and protein content while increasing gel consistence in grains. Metabolome and transcriptome analyses revealed that increased expression of Nhd1 mainly regulates the metabolism of carbohydrates and amino acids in the grain filling stage. Moreover, expression level of Nhd1 shows a positive relationship with grain ECQ in some local main cultivars. Thus, intensifying endogenous abundance of Nhd1 is a promising strategy to upgrade grain ECQ in rice production.

Adaptation to high latitudes through a novel allele of Hd3a strongly promoting heading date in rice.

A novel Hd3a allele strongly promoting rice heading date was identified, and it functions through florigen activation complex (FAC) and was selected during the spread of rice cultivation to high-latitude areas. Heading date is a critical agronomic trait for rice that determines the utilization of light and temperature conditions and thereby affects grain yield. Rice is a short day (SD) plant, and its photoperiodic information is processed by complex pathways and integrated by florigens to control flowering. In this study, we identified a novel allele for the florigen gene Heading date 3a (Hd3a), characterized by a C435G substitution in its coding region, by a genome-wide association study (GWAS) approach in a panel of 199 high-latitude japonica rice varieties. The C435G substitution induces plants to flower 10days earlier in high-latitude area (long day condition). Then, we mutated C435 to G in Hd3a by prime editing and found the point mutation plants flowered 12days earlier. Further molecular experiments showed the novel Hd3a protein can interact with GF14b protein and increase the expression of OsMADS14, the output gene of florigen activation complex (FAC). Molecular signatures of selection indicated that the novel Hd3a allele was selected during the process of rice cultivation expansion into high-latitude areas. Collectively, these results provide new insights into heading date regulation in high-latitude areas and advance improvements to rice adaptability to enhance crop yield.

Natural Variation of OsHd8 Regulates Heading Date in Rice

Heading date, as one of the most important agronomic traits, is a fundamental factor determining crop yield. Although diverse genes related to heading date have already been reported in rice, the key gene that regulates heading date is still poorly understood. Here, we identified a heading date regulator, heading date 8 (OsHd8), which promoted the heading date under long-day conditions and encoded a putative HAP3 subunit of the CCAAT-box-binding transcription factor. It is localized in the nucleus and expressed in various tissues. Sequence analysis revealed that there were four SNPs and one InDel in the promoter region of OsHd8, which was involved in the regulation of some floral regulators including GHD7.1, SDG718, OsGI and HDT1. Further evolutionary analysis showed that OsHd8 presents divergence between indica and japonica, showing natural selection during the domestication of cultivated rice. These results indicate that OsHd8 plays an important role in the regulation of heading date, and may be an important target for rice breeding programs.

Identification and Verification of qGS11, a QTL Controlling Grain Size and Heading Date in Rice

Grain size, shape and weight are important factors influencing grain yield and quality of rice. They are mostly determined by grain length (GL) and grain width (GW). A 13.2 Mb interval, RM167–RM287 on chromosome 11 of rice, was previously found to be associated with variations in 1000-grain weight (TGW). In this study, three populations derived from the indica rice cross Teqing/IRBB52 were used to identify and validate quantitative trait loci (QTLs) controlling GL, GW, TGW and the ratio of GL to GW (RLW) in the RM167–RM287 region. First, two QTL clusters associated with these traits were detected using two populations, segregating the RM167–RM287 interval only. One controlled GL, GW and TGW and was designated as qGS11. The other controlled GL and RLW. The allelic directions of the two QTL clusters on GL were opposite. Then, qGS11 was further mapped in a 1.4 Mb interval using near-isogenic lines, showing a small effect on GL and a relatively large effect on TGW, GW and RLW. Meanwhile, a stable and small effect on the heading date was detected. The allelic direction for the heading date was the opposite for TGW and GW but the same for GL and RLW. The results suggest that qGS11 has the potential for application in rice breeding.

The transcription factor OsGATA6 regulates rice heading date and grain number per panicle.

Heading date, panicle architecture, and grain size are key traits that affect the yield of rice (Oryza sativa). Here, we identified a new gene, OsGATA6, whose product regulates heading date. Overexpression of OsGATA6 resulted in delayed heading, increased grain number, and decreased grain size. Knockdown lines generated by artificial microRNA (amiRNA) and CRISPR genome-edited lines of OsGATA6 both showed earlier heading, decreased grain number, and increased grain size. These results suggested that OsGATA6 negatively regulates heading date, positively regulates panicle development, and affects grain size. OsGATA6 was found to be constitutively expressed in rice, and strongly expressed in young leaves and panicles. In situ hybridization analyses showed that OsGATA6 was specifically localized in superficial cells of the panicle primordium. Overexpression lines show decreased expression of RFT1 and Hd3a, which promote heading. OsMFT1, which delays heading date and increases grain number, was down-regulated in amiRNA lines. Further analyses showed that OsGATA6 could bind to the promoter of OsMFT1 and induce its expression, thereby regulating heading date and panicle development. Overexpression of OsGATA6 in Arabidopsis resulted in repressed expression of AtFT and late flowering, suggesting that its function is similar. Taken together, we have identified a new GATA regulator that influences rice heading date and grain number, which potentially increases rice yield.

Clock component OsPRR59 delays heading date by repressing transcription of Ehd3 in rice

Heading date (or flowering time), an important agronomic trait in crop species, is closely associated with regional adaptation and yield. Members of the Pseudo-Response Regulator (PRR) family play key roles in regulating flowering. However, their role and molecular mechanism controlling heading date in rice is not very clear. Here, we identified rice OsPRR protein, OsPRR59, which delayed heading under long-day conditions. OsPRR59 positively regulates yield by affecting plant height, secondary branches number per panicle, grain number per panicle, seed setting rate, and grain weight per plant. OsPRR59 is expressed in most tissues and its protein is localized to the nucleus. We also found that OsPRR59 directly binds to the promoter of Ehd3 to inhibit its expression. Compared with the WT, osprr59 ehd3 showed a significantly delayed heading phenotype, as did the ehd3 mutant. This was opposite to the phenotype of the osprr59 mutant, confirming that Ehd3 acted downstream of OsPRR59 in regulating rice flowering. Our results identified a direct regulator of Ehd3, and revealed a novel molecular mechanism of clock component OsPRR proteins in regulating heading date and provide a new genetic resource for fine-tuning heading date in rice.

NRL3 Interacts with OsK4 to Regulate Heading Date in Rice

NRL3 is essential for the growth and development of rice leaves. In this study, we found that the loss function of NRL3 also delayed heading date under natural long daylight and short daylight conditions. The yeast two-hybrid and the bimolecular fluorescence complementation proved that NRL3 interacts with OsK4, a Snf1-related kinase. OsK4 localized to the nucleus and expressed in various rice tissues. The rhythmic expression pattern of OsK4 was similar to NRL3 under long daylight and short daylight conditions. Knock-out mutants of OsK4 exhibited early heading under long daylight conditions, indicating that it acts as a negative regulator of heading date in rice. Interestingly, the OsK4 mutant under the nrl3 mutant background rescued the late heading phenotype of nrl3 under long daylight conditions, suggesting that OsK4 functions downstream of NRL3. Moreover, both NRL3 and OsK4 controlled heading date through regulating the expression of Hd3a and RFT1 genes. These findings shed light on the heading date regulation in rice and provide a sound theoretical base to improve regional adaptability of rice.

Predicting Rice Heading Date Using an Integrated Approach Combining a Machine Learning Method and a Crop Growth Model.

Accurate prediction of heading date under various environmental conditions is expected to facilitate the decision-making process in cultivation management and the breeding process of new cultivars adaptable to the environment. Days to heading (DTH) is a complex trait known to be controlled by multiple genes and genotype-by-environment interactions. Crop growth models (CGMs) have been widely used to predict the phenological development of a plant in an environment; however, they usually require substantial experimental data to calibrate the parameters of the model. The parameters are mostly genotype-specific and are thus usually estimated separately for each cultivar. We propose an integrated approach that links genotype marker data with the developmental genotype-specific parameters of CGMs with a machine learning model, and allows heading date prediction of a new genotype in a new environment. To estimate the parameters, we implemented a Bayesian approach with the advanced Markov chain Monte-Carlo algorithm called the differential evolution adaptive metropolis and conducted the estimation using a large amount of data on heading date and environmental variables. The data comprised sowing and heading dates of 112 cultivars/lines tested at 7 locations for 14 years and the corresponding environmental variables (day length and daily temperature). We compared the predictive accuracy of DTH between the proposed approach, a CGM, and a single machine learning model. The results showed that the extreme learning machine (one of the implemented machine learning models) was superior to the CGM for the prediction of a tested genotype in a tested location. The proposed approach outperformed the machine learning method in the prediction of an untested genotype in an untested location. We also evaluated the potential of the proposed approach in the prediction of the distribution of DTH in 103 F2 segregation populations derived from crosses between a common parent, Koshihikari, and 103 cultivars/lines. The results showed a high correlation coefficient (ca. 0.8) of the 10, 50, and 90th percentiles of the observed and predicted distribution of DTH. In this study, the integration of a machine learning model and a CGM was better able to predict the heading date of a new rice cultivar in an untested potential environment.

Evidence for rice Heading date 16 contribution to yield increase under low-nutrient conditions

ABSTRACT Breeding new rice cultivars that are tolerant to low-nutrient condition is a solution to saving the usage of commercial fertilizer. We isolated Hitomebore EMS mutant, 520-6. Seedling growth of 520-6 is tolerant to low-nutrient supply, especially to low phosphorus (low P) supply and genetic mapping revealed the existence of nonsynonymous substitution within Os03g0793500 9th exon in 520-6. Os03g0793500 is reported as Heading date 16 (Hd16)/Early flowering1 (EL1), which is related with the rice flowering pathway. Hd16/EL1 encodes a casein kinase-I protein, and phosphorylates the floral repressor Ghd7 and the rice DELLA protein Slender rice 1 (SLR1). 520-6 carries the same nonfunctional Hd16 allele to that of Koshihikari, and the parental cultivar, Hitomebore, carries the functional Hd16 allele, as with Nipponbare. mRNA accumulation of GA-biosynthesis-related gene, OsGA2ox3 was enhanced in 520-6 compared with Hitomebore, indicating that Hd16 takes a part in feedback regulation of the GA biosynthetic gene. 520-6 and near-isogenic line (NIL) with nonfunctional Hd16 allele showed heavy straw weight and panicle weight in paddy fields with low nitrogen input and no fertilizer application in Kashimadai at Miyagi prefecture and Hikone at Shiga prefecture, respectively. In addition, plants with nonfunctional Hd16 allele showed late flowering phenotype in all tested fields. These results imply that disruption of Hd16 contributes to the improvement of rice growth by positively regulating GA signaling and late flowering, which resulted in increased rice yield under low-nutrient conditions.

The E3 Ubiquitin Ligase HAF1 Modulates Circadian Accumulation of EARLY FLOWERING3 to Control Heading Date in Rice under Long-Day Conditions.

The ubiquitin 26S proteasome system (UPS) is critical for enabling plants to alter their proteomes to integrate internal and external signals for the photoperiodic induction of flowering. We previously demonstrated that HAF1, a C3HC4 RING domain-containing E3 ubiquitin ligase, is essential to precisely modulate the timing of Heading Date1 accumulation and to ensure appropriate photoperiodic responses under short-day conditions in rice (Oryza sativa). However, how HAF1 mediates flowering under long-day conditions remains unknown. In this study, we show that OsELF3 (EARLY FLOWERING3) is the direct substrate of HAF1 for ubiquitination in vitro and in vivo. HAF1 is required for maintaining the circadian rhythm of OsELF3 accumulation during photoperiodic responses in rice. In addition, the haf1 oself3 double mutant headed as late as oself3 plants under long-day conditions. An amino acid variation (L558S) within the interaction domain of OsELF3 with HAF1 greatly contributes to the variation in heading date among japonica rice accessions. The japonica accessions carrying the OsELF3(L)-type allele are found at higher latitudes, while varieties carrying the OsELF3(S)-type allele are found at lower latitudes. Taken together, our findings suggest that HAF1 precisely modulates the diurnal rhythm of OsELF3 accumulation to ensure the appropriate heading date in rice.

QTL mapping for heading date, leaf area and chlorophyll content under cold and drought stress in two related recombinant inbred line populations (Japonica rice) and meta‐analysis

AbstractRice is highly susceptible to drought and cold. The goal of this study was to identify the QTLs affecting the rice heading date (HD), leaf area (LA) and chlorophyll content (CC) under cold and drought stress. A total of twenty‐nine and thirty‐eight additive QTLs were detected from two crosses of ‘Dongnong422’/‘Kongyu131’ and ‘Xiaobaijingzi’/‘Kongyu131’, respectively. qHD1‐7‐1, qHD1‐7‐2, qHD1‐2‐1, qLA1‐7‐1, qLA1‐6‐3 and qCC1‐7‐1 show adaptive effects under cold treatment, while qHD2‐2‐3, qHD2‐2‐2, qLA2‐7‐3 and qCC2‐5‐1 were important for explaining the genetic mechanism during drought tolerance. Additionally, nine and five additive × environment interaction QTLs were detected for two RILs, respectively. RIL26 and RIL73 were two lines that are associated with cold and drought for HD. 339 QTLs related to HD, CC and LA of rice from database and our research were projected onto the genetic map. Nine cloned genes and nineteen homologous candidate genes related to HD, CC, cold tolerance and drought tolerance were mapped by meta‐analysis. These results lay the foundation for the fine mapping of QTLs related to HD, LA and CC for marker‐assisted selection.

Effects of Subsurface Irrigation after the Heading Date of Rice on Homogeneity of Ground Temperature Distribution

Effects of Subsurface Irrigation after the Heading Date of Rice on Homogeneity of Ground Temperature Distribution

The RING-Finger Ubiquitin Ligase HAF1 Mediates Heading date 1 Degradation during Photoperiodic Flowering in Rice.

The photoperiodic response is one of the most important factors determining heading date in rice (Oryza sativa). Although rhythmic expression patterns of flowering time genes have been reported to fine-tune the photoperiodic response, posttranslational regulation of key flowering regulators has seldom been elucidated in rice. Heading date 1 (Hd1) encodes a zinc finger transcription factor that plays a crucial role in the photoperiodic response, which determines rice regional adaptability. However, little is known about the molecular mechanisms of Hd1 accumulation during the photoperiod response. Here, we identify a C3HC4 RING domain-containing E3 ubiquitin ligase, Heading date Associated Factor 1 (HAF1), which physically interacts with Hd1. HAF1 mediates ubiquitination and targets Hd1 for degradation via the 26S proteasome-dependent pathway. The haf1 mutant exhibits a later flowering heading date under both short-day and long-day conditions. In addition, the haf1 hd1 double mutant headed as late as hd1 plants under short-day conditions but exhibited a heading date similar to haf1 under long-day conditions, thus indicating that HAF1 may determine heading date mainly through Hd1 under short-day conditions. Moreover, high levels of Hd1 accumulate in haf1. Our results suggest that HAF1 is essential to precise modulation of the timing of Hd1 accumulation during the photoperiod response in rice.

A nucleotide substitution at the 5′ splice site of intron 1 of rice HEADING DATE 1 (HD1) gene homolog in foxtail millet, broadly found in landraces from Europe and Asia

We investigated genetic variation of a rice HEADING DATE 1(HD1) homolog in foxtail millet. First, we searched for a rice HD1 homolog in a foxtail millet genome sequence and designed primers to amplify the entire coding sequence of the gene. We compared full HD1 gene sequences of 11 accessions (including Yugu 1, a Chinese cultivar used for genome sequencing) from various regions in Europe and Asia, found a nucleotide substitution at a putative splice site of intron 1, and designated the accessions with the nucleotide substitution as carrying a splicing variant. We verified by RT-PCR that this single nucleotide substitution causes aberrant splicing of intron 1. We investigated the geographical distribution of the splicing variant in 480 accessions of foxtail millet from various regions of Europe and Asia and part of Africa by dCAPS and found that the splicing variant is broadly distributed in Europe and Asia. Differences of heading times between accessions with wild type allele of the HD1 gene and those with the splicing variant allele were unclear. We also investigated variation in 13 accessions of ssp. viridis, the wild ancestor, and the results suggested that the wild type is predominant in the wild ancestor.

QTL mapping for thermo-sensitive heading date in rice

The main objective of this study was to dissect the genetic basis of rice flowering regulated by temperature. We used two indica varieties Zhenshan 97 and Zhongzao 18, and 168 recombinant inbred lines (RILs) derived from them to estimate temperature effects on heading date (HD). Tests under four different photoperiod and temperature conditions in growth chambers showed that HD of both parents was accelerated at high temperatures, indicating that both parents were strongly thermo-sensitive. The averaged effective cumulative temperatures (ECTs), 1,120 and 1,263 °C necessary for Zhenshan 97 and Zhongzao 18 respectively, could be the prerequisite in a given condition before heading. QTL analysis was conducted with the RILs grown in both long day and short day conditions in 2 years. A total of 4 ECT QTLs corresponding to 4 HD QTLs were mapped. The major ECT QTL, qEHD10, was repeatedly identified on chromosome 10, which could explain 42.2–57.0 % of ECT variation and 39.8–59.4 % of HD variation in four environments. Overall these novel findings would improve the knowledge of temperature on rice flowering.

QTL Detection and Epistasis Analysis for Heading Date Using Single Segment Substitution Lines in Rice (Oryza sativa L.)

Heading date of rice is a key agronomic trait determining cultivated areas and seasons and affecting yield. In the present study, five primary single segment substitution lines with the same genetic background were used to detect quantitative trait loci (QTLs) for heading date in rice. Two QTLs, qHD3 and qHD6 on the short arm of chromosome 3 and the short arm of chromosome 6, respectively, were identified under natural long-day (NLD). Nineteen secondary single segment substitution lines (SSSLs) and seven double segments pyramiding lines were designed to map the two QTLs and to evaluate their epistatic interaction between them. By overlapping mapping, qHD3 was mapped in a 791-kb interval between SSR markers RM3894 and RM569 and qHD6 in a 1125-kb interval between RM587 and RM225. Results revealed the existence of epistatic interaction between qHD3 and qHD6 under natural long-day (NLD). It was also found that qHD3 and qHD6 had significant effects on plant height and yield traits, indicating that both of the QTLs have pleiotropic effects.

Characterization of Epistatic Interaction of QTLs LH8 and EH3 Controlling Heading Date in Rice

Heading date is a critical trait for adaptation of rice to different cultivation areas and cropping seasons. We evaluated the heading dates of 1,123 chromosome segments substitution lines (CSSLs) in the genetic background of an elite rice variety Huajingxian74 (HJX74). A CSSL with the substituted segments from Zihui100 exhibited late heading under both natural long-day (NLD) and natural short-day (NSD) conditions, and the late heading phenotype was controlled by two novel epistatic loci on chromosome 8 and chromosome 3, respectively, termed LH8 and EH3. The function of EH3 was dependent on the LH8 genotype through epistatic interaction between EH3Zihui100 and LH8Zihui100 alleles. Genetic and molecular characterization revealed LH8 encodes a CCAAT-box-binding transcription factor with Heading date1 (Hd1)-binding activity and may delay flowering by repressing the expression of Early heading date1 (Ehd1). Our work provides a solid foundation for further study on gene interaction in heading date and has application in breeding rice with greater adaptability.

Interaction between temperature and photoperiod in regulation of flowering time in rice

Photoperiod and temperature are two pivotal regulatory factors of plant flowering. The floral transition of plants depends on accurate measurement of changes in photoperiod and temperature. The flowering time of rice (Oryza sativa) as a facultative short-day (SD) plant is delayed under long-day (LD) and/or low temperature conditions. To elucidate the regulatory functions of photoperiod and temperature on flowering time in rice, we systematically analyzed the expression and regulation of several key genes (Hd3a, RFT1, Ehd1, Ghd7, RID1/Ehd2/OsId1, Se5) involved in the photoperiodic flowering regulatory pathway under different temperature and photoperiod treatments using a photoperiod-insensitive mutant and wild type plants. Our results indicate that the Ehd1-Hd3a/RFT1 pathway is common to and conserved in both the photoperiodic and temperature flowering regulatory pathways. Expression of Ehd1, Hd3a and RFT1 is dramatically reduced at low temperature (23°C), suggesting that suppression of Ehd1, Hd3a and RFT1 transcription is an essential cause of delayed flowering under low temperature condition. Under LD condition, Ghd7 mRNA levels are promoted at low temperature (23°C) compared with normal temperature condition (28°C), suggesting low temperature and LD treatment have a synergistic role in the expression of Ghd7. Therefore, upregulation of Ghd7 might be a crucial cause of delayed flowering under low temperature condition. We also analyzed Hd1 regulatory relationships in the photoperiodic flowering pathway, and found that Hd1 can negatively regulate Ehd1 transcription under LD condition. In addition, Hd1 can also positively regulate Ghd7 transcription under LD condition, suggesting that the heading-date of rice under LD condition is also regulated by the Hd1-Ghd7-Ehd1-RFT1 pathway.