Unique miR775-GALT9 module regulates leaf senescence in Arabidopsis during post-submergence recovery by modulating Ethylene and Abscisic acid pathway

Submergence-induced hypoxic condition negatively affects the plant growth and development, and causes early onset of senescence. Hypoxia alters the expression of a number of microRNAs (miRNAs). However, the molecular function of submergence stress-induced miRNAs in physiological or developmental changes and recovery remains poorly understood. Here we show that miR775 is an Arabidopsis thaliana-specific young and unique miRNA that possibly evolved non-canonically. miR775 post-transcriptionally regulates Galactosyltransferase (GALT9) and their expression is inversely affected at 24 hours of complete submergence stress. The overexpression of miR775 (miR775-Oe) confers enhanced recovery from submergence stress and reduced accumulation of RBOHD and ROS, in contrast to wild type and MIM775 Arabidopsis shoot. A similar recovery phenotype of galt9 mutant indicates the role of miR775-GALT9 module in post-submergence recovery. We predicted Golgi-localized GALT9 to be potentially involved in protein glycosylation. The altered expression of senescence-associated genes (SAG12, SAG29, and ORE1), ethylene signalling (EIN2 and EIN3) and ABA biosynthesis (NCED3) pathway genes in miR775-Oe, galt9 and MIM775 plants. Thus, our results indicate the role of miR775-GALT9 module in post-submergence recovery through a crosstalk with ethylene and ABA pathway.

Comprehensive In Silico Analysis and Transcriptional Profiles Highlight the Importance of Mitochondrial Dicarboxylate Carriers (DICs) on Hypoxia Response in Both Arabidopsis thaliana and Eucalyptus grandis

Plant dicarboxylate carriers (DICs) transport a wide range of dicarboxylates across the mitochondrial inner membrane. The Arabidopsis thalianaDIC family is composed of three genes (AtDIC1, 2 and 3), whereas two genes (EgDIC1 and EgDIC2) have been retrieved in Eucalyptus grandis. Here, by combining in silico and in planta analyses, we provide evidence that DICs are partially redundant, important in plant adaptation to environmental stresses and part of a low-oxygen response in both species. AtDIC1 and AtDIC2 are present in most plant species and have very similar gene structure, developmental expression patterns and absolute expression across natural Arabidopsis accessions. In contrast, AtDIC3 seems to be an early genome acquisition found in Brassicaceae and shows relatively low (or no) expression across these accessions. In silico analysis revealed that both AtDICs and EgDICs are highly responsive to stresses, especially to cold and submergence, while their promoters are enriched for stress-responsive transcription factors binding sites. The expression of AtDIC1 and AtDIC2 is highly correlated across natural accessions and in response to stresses, while no correlation was found for AtDIC3. Gene ontology enrichment analysis suggests a role for AtDIC1 and AtDIC2 in response to hypoxia, and for AtDIC3 in phosphate starvation. Accordingly, the investigated genes are induced by submergence stress in A. thaliana and E. grandis while AtDIC2 overexpression improved seedling survival to submergence. Interestingly, the induction of AtDIC1 and AtDIC2 is abrogated in the erfVII mutant that is devoid of plant oxygen sensing, suggesting that these genes are part of a conserved hypoxia response in Arabidopsis.

Improvement of the submergence stress tolerance of local South Sumatran rice through the introgression of the Sub1 gene by using marker-assisted selection

Submergence stress due to unpredictable soil flooding is one of the main constraints encountered in rainfed growing areas, especially in Southern Sumatran riparian swamps. The development of submergence-stress-tolerant cultivars through the introgression of Sub1 via marker-assisted backcrossing (MABC) is an ideal solution. This study was carried out during 2020 at Sriwijaya University, Palembang, Indonesia, with the aim to select Sub1-introgressed lines in BC3F1 generations on the basis of MABC and to screen out the SSR markers that were unlinked to the target gene for application in subsequent background selection studies. Results revealed that almost all the backcrossed progenies segregated from the rice parental cultivars ‘FR13A’ and ‘Pegagan’. The backcrossed lines showed significantly improved submergence stress tolerance and recovery rates compared with their parents. Sub1 introgression into the BC3F1 generation was confirmed by the tightly linked Sub1 marker SUB1C173, and marker RM23915 was used for recombinant selection. These markers followed the expected marker segregation ratio in accordance with the Mendelian single gene model. In the parental polymorphism survey, 84 out of 237 SSR markers that were unlinked to the target loci were found to be available for background study. Twenty-seven backcrossed lines were selected on the basis of foreground selection. Seven plants were selected on the basis of the recombinant marker RM23915. Five backcrossed lines were further selected on the basis of submergence stress tolerance and agronomic performance.

Submergence Gene Sub1A Transfer into Drought-Tolerant japonica Rice DT3 Using Marker-Assisted Selection

Flash flooding is a major environmental stressor affecting rice production worldwide. DT3 is a drought-tolerant, recurrent parent with a good yield, edible quality, and agronomic traits akin to those of an elite Taiwanese variety, Taiken9 (TK9). Progenies carrying Sub1A can enhance submergence stress tolerance and can be selected using the marker-assisted backcross (MAB) breeding method. For foreground selection, Sub1A and SubAB1 were utilized as markers on the BC2F1, BC3F1, and BC3F2 generations to select the submergence-tolerant gene, Sub1A. Background selection was performed in the Sub1A-BC3F2 genotypes, and the percentages of recurrent parent recovery within individuals ranged from 84.7–99.55%. BC3F3 genotypes (N = 100) were evaluated for agronomic traits, yield, and eating quality. Four of the eleven BC3F4 lines showed good yield, yield component, grain, and eating quality. Four BC3F4 lines, SU39, SU40, SU89, and SU92, exhibited desirable agronomic traits, including grain quality and palatability, consistent with those of DT3. These genotypes displayed a high survival rate between 92 and 96%, much better compared with DT3 with 64%, and demonstrated better drought tolerance compared to IR64 and IR96321-345-240. This study provides an efficient and precise MAB strategy for developing climate-resilient rice varieties with good grain quality for flood-prone regions.

Arabidopsis CALMODULIN-LIKE 38 Regulates Hypoxia-Induced Autophagy of SUPPRESSOR OF GENE SILENCING 3 Bodies

During the energy crisis associated with submergence stress, plants restrict mRNA translation and rapidly accumulate stress granules that act as storage hubs for arrested mRNA complexes. One of the proteins associated with hypoxia-induced stress granules in Arabidopsis thaliana is the calcium-sensor protein CALMODULIN-LIKE 38 (CML38). Here, we show that SUPPRESSOR OF GENE SILENCING 3 (SGS3) is a CML38-binding protein, and that SGS3 and CML38 co-localize within hypoxia-induced RNA stress granule-like structures. Hypoxia-induced SGS3 granules are subject to turnover by autophagy, and this requires both CML38 as well as the AAA+-ATPase CELL DIVISION CYCLE 48A (CDC48A). CML38 also interacts directly with CDC48A, and CML38 recruits CDC48A to CML38 granules in planta. Together, this work demonstrates that SGS3 associates with stress granule-like structures during hypoxia stress that are subject to degradation by CML38 and CDC48-dependent autophagy. Further, the work identifies direct regulatory targets for the hypoxia calcium-sensor CML38, and suggest that CML38 association with stress granules and associated regulation of autophagy may be part of the RNA regulatory program during hypoxia stress.

Genome-Wide Identification, Expression and Functional Analysis Reveal the Involvement of FCS-Like Zinc Finger Gene Family in Submergence Response in Rice

Background Direct seeding is an efficient rice cultivation practice. However, its application is often limited due to O2 deficiency following submergence, leading to poor seed germination, seedling establishment, and consequently yield loss. Identification of genes associated with tolerance to submergence and understanding their regulatory mechanisms is the fundamental way to address this problem. Unfortunately, the molecular mechanism of rice response to submergence stress is still not well understood. Results Here, we have performed a genome-wide identification of FCS-like zinc finger (FLZ) proteins and assessed their involvement in submergence response in rice. We identified 29 FLZ genes in rice, and the expression analysis revealed that several genes actively responded to submergence stress. Eight OsFLZ proteins interact with SnRK1A. As a case study, we demonstrated that OsFLZ18 interacted with SnRK1A and inhibited the transcriptional activation activity of SnRK1A in modulating the expression of its target gene αAmy3, a positive regulator in rice flooding tolerance. In line with this, OsFLZ18-overexpression lines displayed retarded early seedling growth and shorter coleoptile following submergence. Conclusions These data provide the most comprehensive information of OsFLZ genes in rice, and highlight their roles in rice submergence response.

Transcriptomic Analysis of the Photosynthetic, Respiration, and Aerenchyma Adaptation Strategies in Bermudagrass (Cynodon dactylon) under Different Submergence Stress

Submergence impedes photosynthesis and respiration but facilitates aerenchyma formation in bermudagrass. Still, the regulatory genes underlying these physiological responses are unclear in the literature. To identify differentially expressed genes (DEGs) related to these physiological mechanisms, we studied the expression of DEGs in aboveground and underground tissues of bermudagrass after a 7 d treatment under control (CK), shallow submergence (SS), and deep submergence (DS). Results show that compared with CK, 12276 and 12559 DEGs were identified under SS and DS, respectively. Among them, the DEGs closely related to the metabolism of chlorophyll biosynthesis, light-harvesting, protein complex, and carbon fixation were down-regulated in SS and DS. Meanwhile, a large number of DEGs involved in starch and sucrose hydrolase activities, glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation were down-regulated in aboveground tissues of bermudagrass in SS and DS. Whereas in underground tissues of bermudagrass these DEGs were all up-regulated under SS, only beta-fructofuranosidase and α-amylase related genes were up-regulated under DS. In addition, we found that DEGs associated with ethylene signaling, Ca2+-ROS signaling, and cell wall modification were also up-regulated during aerenchyma formation in underground tissues of bermudagrass under SS and DS. These results provide the basis for further exploration of the regulatory and functional genes related to the adaptability of bermudagrass to submergence.

Screening Diverse Weedy Rice (Oryza sativa ssp.) Mini Germplasm for Tolerance to Heat and Complete Submergence Stress During Seedling Stage

Rice is a staple food for more than 3.5 billion people worldwide, with Asia producing almost 90% of the global rice yield. In the US, rice is primarily produced in four regions: Arkansas Grand Prairie, Mississippi Delta, Gulf Coast, and Sacramento Valley of California. Arkansas currently accounts for more than 50% of the rice produced in the US. As global temperatures continue to rise and fluctuate, crop-breeding programs must continue to evolve. Unfortunately, sudden submergence due to climate change and unpredictable flash flooding can cause yield reduction up to 100% and affect 20 million ha of agricultural farmlands. Similarly, it has been demonstrated that temperatures higher than 34°C can cause spikelet infertility resulting in up to 60% reduction in yield. One major drawback to developing abiotic stress-tolerant rice is the loss of critical traits such as vegetative vigor, spikelet fertility, and grain quality, which are essential in increasing economic return for farmers. To replace traits lost in past breeding endeavors, weedy rice (WR) has been proposed as a source for novel trait discovery to improve rice breeding programs. Therefore, the goal of this study was to screen and identify heat- and submergence-tolerant WR accessions. A WR mini germplasm consisting of seedlings at the 3–4 leaf stage was exposed to heat (38°C) and complete submergence for 21 days. After each treatment, height was recorded every 7 days for 28 days, and biomass was collected 28 days after treatment. The average height reduction across all accessions was 19 and 21% at 14 and 28 days after treatment (DAT) for the heat-stress treatment. The average height reduction across all accessions was 25 and 33% for the complete submergence stress. The average biomass reduction across all accessions was 18 and 21% for heat and complete submergence stress, respectively. Morphologically, at 28 DAT, 28% of the surviving WR accessions in the heat-stress treatment with <20% height reduction were straw-colored hull types without awns. Under complete submergence stress, 33% of the surviving WR accessions were blackhull types without awns. These specific biotypes may play a role in the increased resilience of WR populations to heat or submergence stress. The results presented in this paper will highlight elite WR accessions that can survive the effects of climate change.

Varied shoot growth in rice plants across different developmental stages under induced flooding

All the modern high yielding rice varieties precariously respond to flooding, although a number of landraces are tolerant to wide range of flooding with penalty of low yield. Stage-specific flood tolerance experiment was performed at three different conditions for three different growth stages from germination to vegetative stage for five such selected landraces, along with a flood-resistant quiescent variety (FR13A), a sensitive line (IR42), an improved cultivar (Swarna) and one Sub1 loci introgressed improved line (Swarna-Sub1). Different morpho-physiological traits at each stage were observed and genotypes were evaluated by these quantifiable traits to understand their underwater performance. All the studied landraces represented strong seed germination and faster coleoptile elongation than FR13A, Swarna and Swarna-Sub1 under water. At early seedling stage var. Kumrogarh embraced with highest number of seedlings with leaves and extended greater leaf portion above the water. Shoot elongation associated with internodal and petiole/blade elongation and well developed aerenchyma tissue facilitated vegetative tissues to survive. In this stage, Kumrogarh had highest plant height but Bakui had highest internodal length which indicated that kumrogarh might have the greater leaf sheath or blade elongation up to day 21 and also smaller increase (%) in air cavity formation at day 21 which made the stem to be upright devoid of lodging. All these results indicated that the studied landraces are the potential resources for submergence avoiding response for all the stages and should be elaborately investigated for future breeding programme. The rice line kumrogarh may be one of the potent traditional rice which can withstand all sorts of submergence by virtue of all the stage-specific attributes under submergence stress.