A transposon insertion in the promoter of OsUBC12 enhances cold tolerance during japonica rice germination.

Low-temperature germination (LTG) is an important agronomic trait for rice (Oryza sativa). Japonica rice generally has greater capacity for germination at low temperatures than the indica subpopulation. However, the genetic basis and molecular mechanisms underlying this complex trait are poorly understood. Here, we report that OsUBC12, encoding an E2 ubiquitin-conjugating enzyme, increases low-temperature germinability in japonica, owing to a transposon insertion in its promoter enhancing its expression. Natural variation analysis reveals that transposon insertion in the OsUBC12 promoter mainly occurs in the japonica lineage. The variation detected in eight representative two-line male sterile lines suggests the existence of this allele introgression by indica-japonica hybridization breeding, and varieties carrying the japonica OsUBC12 locus (transposon insertion) have higher low-temperature germinability than varieties without the locus. Further molecular analysis shows that OsUBC12 negatively regulate ABA signaling. OsUBC12-regulated seed germination and ABA signaling mainly depend on a conserved active site required for ubiquitin-conjugating enzyme activity. Furthermore, OsUBC12 directly associates with rice SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 1.1 (OsSnRK1.1), promoting its degradation. OsSnRK1.1 inhibits LTG by enhancing ABA signaling and acts downstream of OsUBC12. These findings shed light on the underlying mechanisms of UBC12 regulating LTG and provide genetic reference points for improving LTG in indica rice.

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

KEY MESSAGE: 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 10 days 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 12 days 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.

Ubiquitin-Conjugating Enzyme OsUBC11 Affects the Development of Roots via Auxin Pathway.

Rice has 48 ubiquitin-conjugating enzymes, and the functions of most of these enzymes have not been elucidated. In the present study, a T-DNA insertional mutant named R164, which exhibited a significant decrease in the length of primary and lateral roots, was used as the experimental material to explore the potential function of OsUBC11. Analysis using the SEFA-PCR method showed that the T-DNA insertion was present in the promoter region of OsUBC11 gene, which encodes ubiquitin-conjugating enzyme (E2), and activates its expression. Biochemical experiments showed that OsUBC11 is a lysine-48-linked ubiquitin chain-forming conjugase. OsUBC11 overexpression lines showed the same root phenotypes. These results demonstrated that OsUBC11 was involved in root development. Further analyses showed that the IAA content of R164 mutant and OE3 line were significantly lower compared with wild-type Zhonghua11. Application of exogenous NAA restored the length of lateral and primary roots in R164 and OsUBC11 overexpression lines. Expression of the auxin synthesis regulating gene OsYUCCA4/6/7/9, the auxin transport gene OsAUX1, auxin/indole-3-acetic acid (Aux/IAA) family gene OsIAA31, auxin response factor OsARF16 and root regulator key genes, including OsWOX11, OsCRL1, OsCRL5 was significantly down-regulated in OsUBC11 overexpressing plants. Collectively, these results indicate that OsUBC11 modulates auxin signaling, ultimately affecting root development at the rice seedling stage.

First Report of Aphelenchoides besseyi Causing White Tip Disease of Rice in Heilongjiang Province of China.

Aphelenchoides besseyi is considered one of the most damaging plant parasitic nematodes to rice and can cause white tip disease. It was first discovered in Kyushu, Japan in 1915 and introduced into China from Japan in the 1940s. The disease is mainly found in rice-planting areas in South China (Zhu et al. 2016; Wang et al. 2017; Xu et al. 2019), causing yield losses of 30%-50% in irrigated rice (Wang et al. 2014). In summer 2021, rice plants displaying distortion and discoloration on new leaves, white tips on flag leaves, erect and shorter panicles, and fewer or less filled spikelets were observed in the rice field in Acheng district, Harbin city, Heilongjiang Province, China (13°32'33″S, 58°48'51″W). Plant samples with the typical symptoms of 'white tip' in the flag leaves were collected and transported to the laboratory for further pathogen identification. Nematodes were extracted from the leaves and panicles of diseased plants using Baermann funnel method, and the population density of nematode was an average of 57 individuals per sample (total 28 diseased samples). The main morphological characteristics of females (n = 20) and males (n = 15) were individually determined using a microscope (LEICA, DM2500). Female body was slender, and straight to ventrally curved when heat-relaxed, with a rounded cephalic region, the lateral field with four incisures, the excretory pore close to the anterior edge of the nerve ring, a narrow post-vulval sac and less than 1/3 the distance from the vulva to the anus, and a conoid tail with star-shaped mucro. Female measurements: body length = 724.6 (593.3 to 867.5) µm, stylet = 11.7 (10.9 to 12.5) µm, tail length = 39.4 (35.8 to 44.4) µm, a = 45.2 (38.6 to 51.0), b = 10.1 (9.4 to 10.7), c = 18.47 (14.7 to 21.4), and V = 71.1 (65.0 to 79.6). The body of male usually showed in a 'J' shape when relaxed, spicule length between 17.1 and 19.4 µm, and a conoid tail, with terminal mucro bearing two to four pointed processes. Male measurements: body length = 546.3 (521.7 to 587.6) µm, stylet = 11.9 (10.7 to 12.6) µm, tail length = 32.54 (29.53 to 34.35) µm, a = 37.7 (35.7 to 40.4), b = 8.7 (8.5 to 9.1), c = 17.8 (15.8 to19.4). The key morphological characteristics were coincident with the description of A. besseyi by Christie (1942), Ou et al. (2014) and Chen et al. (2020). DNA was extracted from six fresh nematodes, and the partial 18S-rRNA was amplified with a pair of primers 1813F/2646R (5'-CTGCGTGAGAGGTGAAAT-3'; 5'-GCTACCTTGTTACGACTTTT-3') (Holterman et al. 2006). PCR products of expected size were purified and sequenced. The newly obtained sequence (accession no. ON629604) was submitted to GenBank and displayed more than 99.5% identity with that of A. besseyi isolates (KT943536, KT454963, JQ957877). Additionally, the isolated nematodes were further confirmed using the species-specific primers Abess_11F/Abess_11R (5'-GTATTCAATCCCGCGACACT-3'; 5'-CATCCTGTTCGGGCATAGTT-3'), and the PCR amplification generated a 570 bp fragment which was specific to the 28S rDNA region of A. besseyi reported previously (Noronha et al. 2020). The pathogenicity test was confirmed in a pot assay. Approximately 2000 mixed-stage A. besseyi (juveniles, males and females) were obtained from the field-diseased rice plants, and ten-day-old rice seedlings (Japonica rice variety, Keyou 1) (n = 5) were inoculated with 400 nematodes per seedling and maintained in a greenhouse. During early growth stages (fifty days after inoculation), some young leaves of infected plants showed the chlorotic tips. Ninety days after inoculation, similar field symptoms (white tip on leaves) were observed on the aerial tissues of the inoculated plants, and an average of 237 A. besseyi were re-isolated from each plant. To the best of our knowledge, this is the first report of A. besseyi on rice plant in Heilongjiang Province of Northeast China. This finding indicates that A. besseyi could become a severe threat to rice production in Heilongjiang Province and contributes to the further inspection and prevention of white tip disease in rice-growing areas of Heilongjiang Province.

OsASHL1 and OsASHL2, two members of the COMPASS-like complex, control floral transition and plant development in rice.

COMPASS or COMPASS-like is a highly conserved polyprotein complex in eukaryotes that is often involved in methylation of histone H3 lysine 4 (H3K4). However, the biological function of this complex in rice (Oryza sativa) is unclear. Here, we report the identifiction of their functions in growth and development. The osashl1 osashl2 double mutant shows a dwarf and late-flowering phenotype. Lower expression of Ehd1, OsVIL4, and OsMADS51 in the osashl1 osashl2 double mutant background accompanies a delayed vegetative growth phase and photoperiod-sensitive phase compared with that in wild type. Notably, there is less H3K4 mono-, di- and tri-methylation genome-wide in the double mutant, in particular less H3K4 tri-methylation at OsVIL4. Consistent with this result, knockout of OsVIL4 gives rise to a late-flowering phenotype similar to that of the osashl1 osashl2 double mutant, suggesting that OsVIL4 is a target of the COMPASS-like complex. In addition, the expression of key genes in brassinosteroid and gibberellic acid metabolism is altered in the osashl1 osashl2 double mutant, suggesting that the COMPASS-like complex regulates plant growth and development by modulating the levels of these two phytohormones. In summary, we demonstrate that OsASHL1 and OsASHL2 are important for floral transition and plant development.

Differential activity of the antioxidant defence system and alterations in the accumulation of osmolyte and reactive oxygen species under drought stress and recovery in rice (Oryza sativa L.) tillering.

The objective of this study was to investigate the effects of drought stress on the activity of antioxidant enzymes and osmotic adjustment substance content in the tillering period of drought-sensitive and drought-tolerant rice cultivars. The results showed that the superoxide dismutase (SOD), peroxidase (POD), catalase activity (CAT), hydrogen peroxide content, soluble protein content and soluble sugar content increased with the accumulation of time and intensity of drought stress. Compared with the drought-sensitive cultivar, drought-resistant cultivar had a smaller photosynthetic affected area, longer CAT enzyme activity duration, and lower H2O2 accumulation. Unlike POD and CAT enzymes, which maintain the ability to scavenge hydrogen peroxide under long drought conditions, ascorbate peroxidase (APX) enzymes seem to be a rapid response mechanism to scavenge hydrogen peroxide under drought stress. Under a -10 kPa water potential, using soluble sugars on the osmotic adjustment ability of the drought-resistant cultivars was more efficient; under -40 kPa water potential, drought-resistant cultivars can maintain relative high levels of ascorbate (ASA) content in the short term. After the restoration of irrigation, the indices gradually returned to control levels. The ASA content showed faster accumulation ability in drought-resistant cultivars and faster recovery. The soluble protein content recovered more slowly in drought-sensitive cultivars under the -40 kPa treatment. Drought-resistant cultivars showed stronger resistance to drought in the -10 kPa treatment and obtained similar yield to the control, while the drought-sensitive cultivars were more obviously affected by the drought stress.