Rice mutants, selected under severe drought stress, show reduced stomatal density and improved water use efficiency under restricted water conditions.

Introduction: Rice is among the least water-use-efficient crops, and rice plants utilise most of their water uptake for transpirational cooling via stomata. To improve water-use efficiency (WUE) in rice, reducing stomatal density and size could help optimise transpiration and photosynthesis. Methodology: In this study, we compared two series of purple rice stomata mutants: the Stomatal Model Mutant (SMM) identified by microscopic observation of flag-leaf stomata, and the Drought-selected Model Mutant (DMM) generated through screening under severe water stress. After undergoing two rounds of severe water stress between -60 to -80 Ym, right before the R1-2 reproductive stage, three DMMs were selected based on their rapid recovery rate and % filled-grain percentage. Result: The three DMMs displayed 618-697 stomatal units per mm2, similar to the SMMs low-density stomata mutant (JHN 8756 (LD)). Furthermore, the four SMMs, three DMMs and the Jao Hom Nin wild type (JHN WT) were treated with two restricted water condition schemes from seedlings to harvest. The total amount of irrigation and precipitation during the experiment was 78.1 L/plant (69.1 mm/plant) for the less restricted water condition (LR) and 47.5 L/plant (42 mm/plant) for the more restricted water condition (MR). Water condition treatments had no effects on stomatal density and stomatal index. In contrast, genotypes and restricted water condition schemes affected plant height, tillers/plant, % filled grains and shoot dry weight (SDW). The three DMMs and the JHN 8756 (LD), the SMM's low-density stomata mutant, displayed greater resilience towards more restricted water conditions than the SMMs and the JHN wild type. Particularly, DMMs were tolerant to more restricted water condition treatments, showing no SDW penalties. Together, the DMMs and the JHN 8756 (LD) displayed higher WUE under these conditions of more restricted water conditions. Conclusion: A rigorous screening process to distinguish tolerant mutants with a rapid drought recovery rate from severe water stress could pave the way to isolate more mutants with better stomatal functionality and resilience in preparation for imminent climate changes.

Candidate genes affecting stomatal density in rice (Oryza sativa L.) identified by genome-wide association.

Stomata regulate photosynthesis and water loss. They have been an active subject of research for centuries, but our knowledge of the genetic components that regulate stomatal development in crops remains very limited in comparison to the model plant Arabidopsis thaliana. Leaf stomatal density was found to vary by over 2.5-fold across a panel of 235 rice accessions. Using GWAS, we successfully identified five different QTLs associated with stomatal density on chromosomes 2, 3, 9, and 12. Forty-two genes were identified within the haplotype blocks corresponding to these QTLs. Of these, nine genes contained haplotypes that were associated with different stomatal densities. These include a gene encoding a trehalose-6-phosphate synthase, an enzyme that has previously been associated with altered stomatal density in Arabidopsis, and genes encoding a B-BOX zinc finger family protein, a leucine-rich repeat family protein, and the 40 S ribosomal protein S3a, none of which have previously been linked to stomatal traits. We investigated further and show that a closely related B-BOX protein regulates stomatal development in Arabidopsis. The results of this study provide information on genetic associations with stomatal density in rice. The QTLs and candidate genes may be useful in future breeding programs for low or high stomatal density and, consequently, improved photosynthetic capacity, water use efficiency, or drought tolerance.

Functional Bph14 from Rathu Heenati promotes resistance to BPH at the early seedling stage of rice (Oryza sativa L.) as revealed by QTL-seq.

KEY MESSAGE: A QTL associated with BPH resistance at the early seedling stage was identified on chromosome 3. Functional Bph14 in Rathu Heenati was associated with BPH resistance at the early seedling stage. Brown planthopper (BPH; Nilaparvata lugens Stål) is considered the most important rice pest in many Asian countries. Several BPH resistance genes have previously been identified. However, there are few reports of genes specific for BPH resistance at the early seedling stage, a crucial stage for direct-seeding cultivation. In this study, we performed a QTL-seq analysis using two bulks (20 F2 lines in each bulk) of the F2 population (n = 300) derived from a cross of Rathu Heenati (RH) × HCS-1 to identify QTL/genes associated with BPH resistance at the early seedling stage. An important QTL was identified on chromosome 3 and Bph14 was identified as a potential candidate gene based on the differences in gene expression and sequence variation when compared with the two parents. All plants in the resistant bulks possessed the functional Bph14 from RH and all plants in the susceptible bulk and HCS-1 contained a large deletion (2703 bp) in Bph14. The functional Bph14 gene of RH appears to be important for BPH resistance at the early seedling stage of rice and could be used in conjunction with other BPH resistance genes in rice breeding programs that confer resistance to BPH at the early and later growth stages.

Induced Genetic Variations in Stomatal Density and Size of Rice Strongly Affects Water Use Efficiency and Responses to Drought Stresses.

Rice (Oryza sativa L.) is an important food crop relied upon by billions of people worldwide. However, with increasing pressure from climate change and rapid population growth, cultivation is very water-intensive. Therefore, it is critical to produce rice that is high-yielding and genetically more water-use efficient. Here, using the stabilized fast-neutron mutagenized population of Jao Hom Nin (JHN) - a popular purple rice cultivar - we microscopically examined hundreds of flag leaves to identify four stomatal model mutants with either high density (HD) or low density (LD) stomata, and small-sized (SS) or large-sized (LS) stomata. With similar genetic background and uniformity, the stomatal model mutants were used to understand the role of stomatal variants on physiological responses to abiotic stress. Our results show that SS and HD respond better to increasing CO2 concentration and HD has higher stomatal conductance (gs) compared to the other stomatal model mutants, although the effects on gas exchange or overall plant performance were small under greenhouse conditions. In addition, the results of our drought experiments suggest that LD and SS can better adapt to restricted water conditions, and LD showed higher water use efficiency (WUE) and biomass/plant than other stomatal model mutants under long-term restricted water treatment. Finally, our study suggests that reducing stomata density and size may play a promising role for further work on developing a climate-ready rice variety to adapt to drought and heat stress. We propose that low stomata density and small size have high potential as genetic donors for improving WUE in climate-ready rice.

A SNP of betaine aldehyde dehydrogenase (BADH) enhances an aroma (2-acetyl-1-pyrroline) in sponge gourd (Luffa cylindrica) and ridge gourd (Luffa acutangula).

Luffa is a genus of tropical and subtropical vines belonging to the Cucurbitaceae family. Sponge gourd (Luffa cylindrica) and ridge gourd (Luffa acutangula) are two important species of the genus Luffa and are good sources of human nutrition and herbal medicines. As a vegetable, aromatic luffa is more preferred by consumers than nonaromatic luffa. While the aroma trait is present in the sponge gourd, the trait is not present in the ridge gourd. In this study, we identified Luffa cylindrica's betaine aldehyde dehydrogenase (LcBADH) as a gene associated with aroma in the sponge gourd based on a de novo assembly of public transcriptome data. A single nucleotide polymorphism (SNP: A > G) was identified in exon 5 of LcBADH, causing an amino acid change from tyrosine to cysteine at position 163, which is important for the formation of the substrate binding pocket of the BADH enzyme. Based on the identified SNP, a TaqMan marker, named AroLuff, was developed and validated in 370 F2 progenies of the sponge gourd. The marker genotypes were perfectly associated with the aroma phenotypes, and the segregation ratios supported Mendelian's simple recessive inheritance. In addition, we demonstrated the use of the AroLuff marker in the introgression of LcBADH from the aromatic sponge gourd to the ridge gourd to improve aroma through interspecific hybridization. The marker proved to be useful in improving the aroma characteristics of both Luffa species.

Primary Root Excision Induces ERF071, Which Mediates the Development of Lateral Roots in Makapuno Coconut (Cocos nucifera).

Coconut (Cocos nucifera L.) is widely recognized as one of nature's most beneficial plants. Makapuno, a special type of coconut with a soft, jelly-like endosperm, is a high-value commercial coconut and an expensive delicacy with a high cost of planting material. The embryo rescue technique is a very useful tool to support mass propagation of makapuno coconut. Nevertheless, transplanting the seedlings is a challenge due to poor root development, which results in the inability of the plant to acclimatize. In this study, primary root excision was used in makapuno to observe the effects of primary root excision on lateral root development. The overall results showed that seedlings with roots excised had a significantly higher number of lateral roots, and shoot length also increased significantly. Using de novo transcriptome assembly and differential gene expression analysis, we identified 512 differentially expressed genes in the excised and intact root samples. ERF071, encoding an ethylene-responsive transcription factor, was identified as a highly expressed gene in excised roots compared to intact roots, and was considered a candidate gene associated with lateral root formation induced by root excision in makapuno coconut. This study provides insight into the mechanism and candidate genes involved in the development of lateral roots in coconut, which may be useful for the future breeding and mass propagation of makapuno coconut through tissue culture.

Identification and Validation of a QTL for Bacterial Leaf Streak Resistance in Rice (Oryza sativa L.) against Thai Xoc Strains.

Rice is one of the most important food crops in the world and is of vital importance to many countries. Various diseases caused by fungi, bacteria and viruses constantly threaten rice plants and cause yield losses. Bacterial leaf streak disease (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most devastating rice diseases. However, most modern rice varieties are susceptible to BLS. In this study, we applied the QTL-seq approach using an F2 population derived from the cross between IR62266 and Homcholasit (HSC) to rapidly identify the quantitative trait loci (QTL) that confers resistance to BLS caused by a Thai Xoc isolate, SP7-5. The results showed that a single genomic region at the beginning of chromosome 5 was highly associated with resistance to BLS. The gene xa5 was considered a potential candidate gene in this region since most associated single nucleotide polymorphisms (SNPs) were within this gene. A Kompetitive Allele-Specific PCR (KASP) marker was developed based on two consecutive functional SNPs in xa5 and validated in six F2 populations inoculated with another Thai Xoc isolate, 2NY2-2. The phenotypic variance explained by this marker (PVE) ranged from 59.04% to 70.84% in the six populations. These findings indicate that xa5 is a viable candidate gene for BLS resistance and may help in breeding programs for BLS resistance.

Rice Stomatal Mega-Papillae Restrict Water Loss and Pathogen Entry.

Rice (Oryza sativa) is a water-intensive crop, and like other plants uses stomata to balance CO2 uptake with water-loss. To identify agronomic traits related to rice stomatal complexes, an anatomical screen of 64 Thai and 100 global rice cultivars was undertaken. Epidermal outgrowths called papillae were identified on the stomatal subsidiary cells of all cultivars. These were also detected on eight other species of the Oryza genus but not on the stomata of any other plant species we surveyed. Our rice screen identified two cultivars that had "mega-papillae" that were so large or abundant that their stomatal pores were partially occluded; Kalubala Vee had extra-large papillae, and Dharia had approximately twice the normal number of papillae. These were most accentuated on the flag leaves, but mega-papillae were also detectable on earlier forming leaves. Energy dispersive X-Ray spectrometry revealed that silicon is the major component of stomatal papillae. We studied the potential function(s) of mega-papillae by assessing gas exchange and pathogen infection rates. Under saturating light conditions, mega-papillae bearing cultivars had reduced stomatal conductance and their stomata were slower to close and re-open, but photosynthetic assimilation was not significantly affected. Assessment of an F3 hybrid population treated with Xanthomonas oryzae pv. oryzicola indicated that subsidiary cell mega-papillae may aid in preventing bacterial leaf streak infection. Our results highlight stomatal mega-papillae as a novel rice trait that influences gas exchange, stomatal dynamics, and defense against stomatal pathogens which we propose could benefit the performance of future rice crops.