Loss of OsMATE6 Function Enhances Drought Resistance Without Yield Penalty by Regulating Stomatal Closure in Rice.

Drought is one of the most severe environmental factors limiting plant growth and crop yield, necessitating the identification of genes that enhance drought resistance for crop improvement. Through screening an ethyl methyl sulfonate-mutagenized rice mutant library, we isolated the PEG tolerance mutant 97-1 (ptm97-1), which displays enhanced resistance to osmotic and drought stress, and increased yield under drought conditions. A point mutation in OsMATE6 was identified as being associated with the drought-resistant phenotype of ptm97-1. The role of OsMATE6 in conferring drought resistance was confirmed by additional OsMATE6 knockout mutants. OsMATE6 is expressed in guard cells, shoots and roots and the OsMATE6-GFP fusion protein predominantly localizes to the plasma membrane. Our ABA efflux assays suggest that OsMATE6 functions as an ABA efflux transporter; mutant protoplasts exhibited a slower ABA release rate compared to the wild type. We hypothesize that OsMATE6 regulates ABA levels in guard cells, influencing stomatal closure and enhancing drought resistance. Notably, OsMATE6 knockout mutants demonstrated greater yields under field drought conditions compared to wild-type plants, highlighting OsMATE6 as a promising candidate for improving crop drought resistance.

OsNLP3 enhances grain weight and reduces grain chalkiness in rice.

Grain weight, a key determinant of yield in rice (Oryza sativa L.), is governed primarily by genetic factors, whereas grain chalkiness, a detriment to grain quality, is intertwined with environmental factors such as mineral nutrients. Nitrogen (N) is recognized for its effect on grain chalkiness, but the underlying molecular mechanisms remain to be clarified. This study revealed the pivotal role of rice NODULE INCEPTION-LIKE PROTEIN 3 (OsNLP3) in simultaneously regulating grain weight and grain chalkiness. Our investigation showed that loss of OsNLP3 leads to a reduction in both grain weight and dimension, in contrast to the enhancement observed with OsNLP3 overexpression. OsNLP3 directly suppresses the expression of OsCEP6.1 and OsNF-YA8, which were identified as negative regulators associated with grain weight. Consequently, two novel regulatory modules, OsNLP3-OsCEP6.1 and OsNLP3-OsNF-YA8, were identified as key players in grain weight regulation. Notably, the OsNLP3-OsNF-YA8 module not only increases grain weight but also mitigates grain chalkiness in response to N. This research clarifies the molecular mechanisms that orchestrate grain weight through the OsNLP3-OsCEP6.1 and OsNLP3-OsNF-YA8 modules, highlighting the pivotal role of the OsNLP3-OsNF-YA8 module in alleviating grain chalkiness. These findings reveal potential targets for simultaneous enhancement of rice yield and quality.

Rice NIN-LIKE PROTEIN 3 modulates nitrogen use efficiency and grain yield under nitrate-sufficient conditions.

Nitrogen (N) is an essential macronutrient for crop growth and yield. Improving the N use efficiency (NUE) of crops is important to agriculture. However, the molecular mechanisms underlying NUE regulation remain largely elusive. Here we report that the OsNLP3 (NIN-like protein 3) regulates NUE and grain yield in rice under N sufficient conditions. OsNLP3 transcript level is significantly induced by N starvation and its protein nucleocytosolic shuttling is specifically regulated by nitrate. Loss-of-function of OsNLP3 reduces plant growth, grain yield, and NUE under sufficient nitrate conditions, whereas under low nitrate or different ammonium conditions, osnlp3 mutants show no clear difference from the wild type. Importantly, under sufficient N conditions in the field, OsNLP3 overexpression lines display improved grain yield and NUE compared with the wild type. OsNLP3 orchestrates the expression of multiple N uptake and assimilation genes by directly binding to the nitrate-responsive cis-elements in their promoters. Overall, our study demonstrates that OsNLP3, together with OsNLP1 and OsNLP4, plays overlapping and differential roles in N acquisition and NUE, and modulates NUE and the grain yield increase promoted by N fertilizer. Therefore, OsNLP3 is a promising candidate gene for the genetic improvement of grain yield and NUE in rice.

Suppression of rice miR168 improves yield, flowering time and immunity.

MicroRNA168 (miR168) is a key miRNA that targets Argonaute1 (AGO1), a major component of the RNA-induced silencing complex1,2. Previously, we reported that miR168 expression was responsive to infection by Magnaporthe oryzae, the causal agent of rice blast disease3. However, how miR168 regulates immunity to rice blast and whether it affects rice development remains unclear. Here, we report our discovery that the suppression of miR168 by a target mimic (MIM168) not only improves grain yield and shortens flowering time in rice but also enhances immunity to M. oryzae. These results were validated through repeated tests in rice fields in the absence and presence of rice blast pressure. We found that the miR168-AGO1 module regulates miR535 to improve yield by increasing panicle number, miR164 to reduce flowering time, and miR1320 and miR164 to enhance immunity. Our discovery demonstrates that changes in a single miRNA enhance the expression of multiple agronomically important traits.

Rice NIN-LIKE PROTEIN 4 plays a pivotal role in nitrogen use efficiency.

Nitrogen (N) is one of the key essential macronutrients that affects rice growth and yield. Inorganic N fertilizers are excessively used to boost yield and generate serious collateral environmental pollution. Therefore, improving crop N use efficiency (NUE) is highly desirable and has been a major endeavour in crop improvement. However, only a few regulators have been identified that can be used to improve NUE in rice to date. Here we show that the rice NIN-like protein 4 (OsNLP4) significantly improves the rice NUE and yield. Field trials consistently showed that loss-of-OsNLP4 dramatically reduced yield and NUE compared with wild type under different N regimes. In contrast, the OsNLP4 overexpression lines remarkably increased yield by 30% and NUE by 47% under moderate N level compared with wild type. Transcriptomic analyses revealed that OsNLP4 orchestrates the expression of a majority of known N uptake, assimilation and signalling genes by directly binding to the nitrate-responsive cis-element in their promoters to regulate their expression. Moreover, overexpression of OsNLP4 can recover the phenotype of Arabidopsis nlp7 mutant and enhance its biomass. Our results demonstrate that OsNLP4 plays a pivotal role in rice NUE and sheds light on crop NUE improvement.

Overexpression of Arabidopsis NLP7 improves plant growth under both nitrogen-limiting and -sufficient conditions by enhancing nitrogen and carbon assimilation.

Nitrogen is essential for plant survival and growth. Excessive application of nitrogenous fertilizer has generated serious environment pollution and increased production cost in agriculture. To deal with this problem, tremendous efforts have been invested worldwide to increase the nitrogen use ability of crops. However, only limited success has been achieved to date. Here we report that NLP7 (NIN-LIKE PROTEIN 7) is a potential candidate to improve plant nitrogen use ability. When overexpressed in Arabidopsis, NLP7 increases plant biomass under both nitrogen-poor and -rich conditions with better-developed root system and reduced shoot/root ratio. NLP7-overexpressing plants show a significant increase in key nitrogen metabolites, nitrogen uptake, total nitrogen content, and expression levels of genes involved in nitrogen assimilation and signalling. More importantly, overexpression of NLP7 also enhances photosynthesis rate and carbon assimilation, whereas knockout of NLP7 impaired both nitrogen and carbon assimilation. In addition, NLP7 improves plant growth and nitrogen use in transgenic tobacco (Nicotiana tabacum). Our results demonstrate that NLP7 significantly improves plant growth under both nitrogen-poor and -rich conditions by coordinately enhancing nitrogen and carbon assimilation and sheds light on crop improvement.

[Genetic analysis of dense and erect panicle 2 allele DEP2-1388 and its application in hybrid rice breeding].

Using ethyl methanesulfonate (EMS) mutagenesis, we isolated an erect panicle mutant, R1338, from indica heavy-panicle restorer Shuhui498. Compared with wild type control, the mutant displayed dwarfism, erect and short panicle, short primary panicle branch, increased grain density, short grain length and increased grain thickness. In addition, the erect panicle architecture of R1388 resulted in significant decreased bending moment and increased resistance to panicle bending. Histocytological analysis indicated that the diameter of uppermost internode, cellulose content and lignin content play important roles in resistance to panicle bending. Genetic analysis revealed that the mutant phenotype was controlled by a semi-dominant nuclear gene. With resequencing and MutMap analysis strategy, we found that one SNP from A to G at the seventh exon of DEP2 resulted in the 928(th) amino acid substitution from arginine (AGG) to glycine (GGG) in R1338 mutant. Considering the phenotype of other dep2 mutants, the phenotype of R1338 was likely to be caused by the SNP in DEP2. The mutant R1338 and wild type were crossed with several sterile lines which respectively had different panicle types, the combinations generated from R1338 and curve panicle sterile lines showed semi-erect panicle, higher seed setting percentage and heterosis, and the combinations generated from R1388 and erect panicle sterile line with DEP1 showed erect panicle by gene additive effect. Moreover, the combinations with semi-erect panicle had superior light transmittance and stronger light intensity, which improved efficiency of light utilization to intermediate and subjacent leaves compared to the combinations with curved panicle. This study provides a good strategy to solve the problem of population density in three-line hybrid rice.

[Effects of Trichoderma longbrachitum and Streptomyces jingyangensis combination on the growth and disease resistance of tobacco seedlings].

An agar plate antagonism experiment in combining with in vivo screening experiment was conducted to study the affinity and bacteriostasis spectrum of the combination of biocontrol agents Trichoderma longbrachitum and Streptomyces jingyangensis to Nicotiana tabacum seedlings, with the effects of each agent and their combination on the N. tabacum seedlings growth, induced resistance, and resistance to Phytophthora nicotianae analyzed. The two agents had no interactive inhibitory effect and showed higher affinity to N. tabacum, and the agents themselves as well as their metabolites had higher bacteriostasis activities and wider bacteriostasis spectrum to P. nicotiaonae, Pythium aphanidermatum, and Alternaria alternate in different habitats. The combination of the two agents affected the morphological characteristics of the seedlings underground and aboveground parts, promoted the growth of root, stem, and leaf, and increased the root volume, total surface area, length, and average diameter as well as the stem height and size and the leaf length, width, and biomass, with these promotion effects being superior than those of the single-agent treatment. The combination of the two agents also increased the activities of the defensive enzymes superoxide dismutase, catalase, phenylalanine ammonia lyase, and peroxidase in the seedlings root significantly, with the relative control efficiency against P. nicotianae reached 69.3%, as compared to the conventional treatment. This study showed that the combination of T. longbrachitum and S. jingyangensis was a compatible combination with higher affinity and efficiency. This combination showed a synergistic effect of the two agents in plant disease control and in promoting plant growth, being able to promote the tobacco seedlings growth and control the P. nicotianae effectively.

[Genetic analysis and mapping of a rice white midrib mutant Oswm2].

A rice (Oryza sativa L.) plant with white midrib (Oswm2) was selected from the T-DNA inserted mutant pool of rice. The basal midrib of the leaves in the mutant, except for the flag leaves, is white. The blade close to the white midrib is etiolated, and alterations of the agronomic traits, such as plant height, panicle length occur in the mutant. Genetic analysis indicated that this mutant trait was controlled by a recessive nuclear gene. To map the OsWM2 gene, an F2 population was constructed by crossing mutant Oswm2 with 02428. The OsWM2 locus was preliminarily located between the SSR molecular markers RM21478 and RM418 on chromosome 7 with the distances of 8.7 and 15.9 cM, respectively.

[Genetic diversity of AVR-pita alleles of rice blast fungus Magnaporthe grisea].

Rice blast, caused by fungus Magnaporthe grisea, is one of the most serious diseases of rice throughout the world and is also a classical system in which avirulence (AVR) genes in the pathogen show a functional correspondence with particular resistance (R) genes in rice. The interaction between AVR-pita gene in Magnaporthe grsiea and Pi-ta gene in rice is a classical gene-for-gene system. The genetic diversity of AVR-pita alleles was showed by 44 isolates from the fields in Sichuan and Chongqing. According to the published sequence of AVR-pita (GenBank Accession No. AF207841), the special primers were designed and a population analysis of AVR-pita occurrence in the field was conducted. The PCR negative isolates did not trigger host resistance responses and the disease might occur. The positive special PCR band in an isolate was not corresponding with its avirulence to the Pi-ta rice line. Among 11 sequenced A VR-pita alleles, six AVR-pita alleles encoded the same protein, four proteins differed from each other by one amino acid and one had nonsense mutation. The strategy of utilizing Pi-ta in rice breeding was also discussed.

[PTD-hEGF fusion protein--gene expression and function analysis].

To produce membrane-permeable human epidermal growth factor (hEGF), a pPTD-hEGF prokaryocyte expression vector was constructed and transformed into E. coli BL 21 (DE3). The PTD-hEGF fusion protein was induced by 0.3 mmol/L of IPTG expressed in the form of inclusion body with an yield of 40% of the total protein in the cells, and then purified by Ni2+ -NTA affinity chromatography. The SDS-PAGE analysis showed a single fusion protein band with a molecular weight of 16 kD. The amino acid sequence was checked by MALDI-TOF-MS analysis. The genetic engineering PTD-hEGF fusion protein obviously promoted the proliferation and growth of the HEK-293 cells in vitro.

Ultrastructure and gene mapping of the albino mutant al12 in rice (Oryza sativa L.).

Seedling albino mutation resistant to low temperature is an adaptability of rice (Oryza sativa L.) to cold. The mutant, a conditional expression controlled by development and temperature, differs from other albino mutants. The chlorophyll content of the mutant was measured using a portable chlorophyll meter, and the ultrastructure of the chloroplast was observed using a transmission electron microscope. Chlorophyll content was 1.2 SPAD, and the chloroplast did not develop, with only small vesicle-like structures. A segregation analysis of the reciprocal crosses between the albino mutation line with the rice line 9311 demonstrated that the albino trait was controlled by a single recessive gene, which was flanked by SSR markers RM5068 and RM3702 on the short arm of chromosome 8 with a distance of 0.5-1.1 cM and 4.9 cM, respectively. This gene was mapped within a 6 cM interval region and was tentatively referred to as al12.

[Morphological and anatomical characterization of a stripe mutant with abnormal floral organs in rice].

A rice double mutant was derived from the transgenic process,but it does not carry the alien gene. The mutant showed white stripe on stem, leaf and spikelet. In some growing stage,the leaf started to produce fork or curliness. The floret number increased, showing multi-lemma/palea, palea-like or lemma-like lodicules or enlarged lodicules, additional pistil and stamen and the spited floret. With observation of cell ultra structure using electron microscope,the white tissue showed concaved cell wall and abnormal plastid which could not develop normal lamellae and thylakoid. The contents of chlorophyll and net photosynthesis rate in the mutant were obviously lower than those in the wild type. The cells in green sectors grow normally with the exception of the bigger cell volume. The morphogenesis of floral organ was observed by using the scanning electron microscopy (SEM). Results showed that the stamen development was not synchronal and the sizes of stamen primordium were different in mutant, and the carpel was smaller than that of wild type.

Genetic analysis and physical mapping of Lk-4(t), a major gene controlling grain length in rice, with a BC2F2 population.

Grain size and shape are important factors affecting grain quality and yield in rice. Mapping, tagging and identification of their related genes can lead us to understand their expression pattern and mechanism network, which is to their control. In this study we mapped a grain length controlling gene named Lk-4(t) with SSR and CAPs markers by screening 800 recessive plants in a BC2F2 population derived from a cross of Shuhui527xXiaoli and backcrossed with Xiaoli as the donor parent. The distribution of grain shape parameters and thousand grain weight in F2 and BC2F2 population showed that backcross can diminish most unnecessary variations to identify the target gene more clearly. There were only two grain length phenotypes found among the 3 209 BC2F2 plants, long and short, indicating it is a qualitative trait. The frequency distribution for the grain length showed a typical segregation ratio of 3:1, suggesting that only one allele was responsible for the variation. By screening the recessive long grain plants with three CAPs markers, P1-EcoRV, P2-Sac I and P3-Mbo I, we tagged the locus on the arm of chromosome 3 near the centromere. Lk-4(t) was located between P1- EcoRV and P2-Sac I, with genetic distance of 0.90 cM and 0.50 cM from the two markers respectively. Mapping of the gene is a foundation for its final identification and function analysis.

[SSR markers linked with early stability in rice].

One hundred and thirty hybrids derived from the crosses between nine rice strains stabilized in early generation and seven cultivars (Oryza sativa L.). In their F2 populations, 32 uniform strains of different agronomic traits were observed. In the same combination of these uniform strains,there were strains segregating in the Mendelian manner. SSR markers analysis showed that F2 and F3 populations of the uniform strains and their F1 plants displayed the same markers indicating all the uniform strains were homozygous. All the marker bands of parents were simultaneously present on their progeny chromosomes, indicating that the uniform strains must true hybrid origin. The SSR markers of F2 population of segregating strains and their F1 plants showed that the segregating strains were heterzygosity. SSR marker band sites of RM276, RM258, RM248 and RM1 loci in most uniform strains presented at high frequencies bands site different from their parent's. We suggested that during the gamete formation,the genes that linked to ga-1, Rf and ga-8 mutated at certain frequency,and followed by a somatic meiosis of the fecundated zygote. Thus a haploid cell may develop into a homozygous embryo,and the F1 plant was homozygous and the progeny stabilized in early generation.

[Study on early stability by ISSR markers in rice].

In the F2 population crossed from two early stability rice (Oryza sativa L.) with four cultivars, eight uniform strains were recorded. Genetic analysis showed that both uniform strains with uniform agronomic characteristics and segregated strains segregating in Mendelian manner were observed in F2 population of the same combination. Four types of marker-bands were obtained after the F2 uniform strains were marked by 26 ISSR primers:t ype I, maternal marker bands present, and paternal marker bands absent; type II, paternal marker bands present, and maternal marker bands absent; type III, parts of maternal and paternal marker bands present, and the others absent; type IV, new recombined marker bands appeared, and all maternal and paternal marker bands absent. But segregating strains showed heterzygosity marker bands, maternal and paternal marker bands being present. The uniform strains and normal strains in rice might be grouped into two classes on the basis of the 2000 bp marker band amplified by the ISSR marker primers No. 900. This result would provide experimental basis for the study on genetic mechanism of early stability in rice.

[Cloning and analysis of a new NBS-LRR resistance gene family in rice].

Sequence-based gene isolation has been a practical approach for plant resistance gene cloning. In this study, RS13, a cloned rice sequence with the NBS (nucleotide-binding site) domain of resistance genes, was used as a probe to screen a bacterial artificial chromosome (BAC) library of rice variety IR64,and four positive clones were obtained. Of them the clone 14E19 covered the other three clones and was sequenced through a shotgun approach. The whole sequence of the insert fragment of 14E19 was assembled into approximately 73 kb in length. Genes on the whole assembled sequence were predicted,and four genes encoding NBS and LRR (leucine-rich repeat) domains were found, named as NL-A, B, C and D respectively. For further analysis, another longer BAC clone,106P13, covering 14E19 on the same chromosome position was identified from a BAC library of IRBB56 which had the same genome background with IR64. Ten NL-homologous copies were discovered on the sequence of the BAC clone 106P13, and four copies were identical with those on 14E19. The similar homologous sequences were also found in the genomic sequences of Nipponbare,93-11 and Guangluai4. However, NL sequences were less homologous with the known NBS-LRR resistance genes. This result indicated that NL was a new NBS-LRR gene family and was composed of ten members at least. RT-PCR and cDNA screening displayed that NL-B expressed in a bacterial blight-resistant rice variety IRBB4, indicating the gene was possibly involved in resistance reactions.

[Generation and identification of rice T-DNA insertional mutant lines].

By using the matured embryos of Japonica rice variety Zhonghua No. 11 as explants, rice transformation was performed by Agrobacterium-mediated co-cultivation method, resulting in 1489 independent transgenic rice plants that carry a T-DNA insertion. Genomic DNA gel-blot and PCR analyses showed that 69.8% of the total lines contain the inserted T-DNA. The flanking sequence of T-DNA in transgenic rice plants was analyzed using Tail-PCR. In addition, we have evaluated 1066 T1 transgenic lines on heading days, plant height and panicles per hill, and found different types of mutants from a number of lines.

[Marker-assisted selection and pyramiding for three blast resistance genes, Pi-d(t)1, Pi-b, Pi-ta2, in rice].

G46B is a promising holding line used for three-lines breeding strategy in hybrid rice, but it is susceptible to blast disease caused by Pyricularia grisea. To improve its blast resistance, three rice varieties, Digu, BL-1, and Pi-4, with blast resistance genes, Pi-d(t), Pi-b, and Pi-ta2, respectively, were used to be crossed with G46B, and 15 plants with these three blast resistance genes, Pi-d(t)1, Pi-b, and Pi-ta2, were selected from their F2 and B1C1 populations via a marker-aided crossing procedure. Among them, four plants were heterozygotes in the three resistance genes, with the genotype of Pi-d(t)1 pi-d(t)/Pi-b pi-b/ Pi-ta2 pi-ta2; ten plants were heterozygotes in two of the three resistance genes, of which six with the genotype of Pi-d(t)1 Pi-d(t)1/Pi-b pi-b/Pi-ta2 pi-ta2, three with the genotype of Pi-d(t)1 pi-d(t)1/Pi-b pi-b/Pi-ta2 Pi-ta2, and one with the genotype of Pi-d(t)1pi-d(t)1/Pi-b Pi-b/Pi-ta2 pi-ta2; and only one plant was homozygote in two of the three resistance genes with the genotype of Pi-d(t)1 Pi-d(t)/Pi-b pi-b/Pi-ta2 Pi-ta2. These results demonstrate the capacity of maker-assisted selection (MAS) in gene pyramiding for rice blast resistance and its enhancement for the efficiency in rice resistance breeding.

[Accelerated improvement of bacterial blight resistance of 'Shuhui527' using molecular marker-assisted selection].

'Shuhui527' is a promising restorer line bred by Rice Research Institute of Sichuan Agricultural University in recent years. However, this line is susceptible to Bacterial Blight (BB), which limits its use. The IRBB60, from the International Rice Research Institute (IRRI), contains dominant genes Xa21 and Xa4 conferring resistance to BB. The objective of this study is to improve the BB resistance of 'Shuhui527' by introgressing Xa21 and Xa4, the two broad-spectrum BB resistance genes, into 'Shuhui527' with IRBB60 as the donor, pTA248 and MP12, linking tightly with Xa21 and Xa4 respectively as DNA markers. BC1 F1 progenies of (Shuhui527 x IRBB60), containing Xa21 and Xa4 identified using PCR screening and with agronomic traits including plant type, grain type and days to heading etc similar to those of 'Shuhui527', were subsequently backcrossed to 'Shuhui527' and self-pollinated to generate BC2 F1 and BC1 F2 . The BC3 F1 and BC3 F2 were subsequently developed using the same approach. Among the 20 BC3 F2 plants, homozygous Xa21 and Xa4,10 plants were the most similar to 'Shuhui527' in the agronomic traits, and were screened using 120 pairs SSR and 100 pairs RAPD markers. Based on the results of the background screening and the performance of the agronomic traits, 5 plants were identified as improved-'Shuhui527' and designated as 527R-5, 527R-6, 527R-8, 527R-9 and 527R-10. The improved-' Shuhui527' lines expressed high resistance to Xanthomonas oryzae pv . oryzae (Xoo) stains C I - C VII, P1 and P6. The evaluation of the polymorphisms and selection accuracies of pTA248 and MP12 demonstrated that the polymorphisms of the two markers were obvious and co-dominant and the accuracies were more than 97% and 83% respectively, indicating the two markers are good for Xa21 and Xa4 in Molecular Marker-assisted Selection.