Rapid improvement of grain appearance in three-line hybrid rice via CRISPR/Cas9 editing of grain size genes.

KEY MESSAGE: Genetic editing of grain size genes quickly improves three-line hybrid rice parents to increase the appearance quality and yield of hybrid rice. Grain size affects rice yield and quality. In this study, we used CRISPR/Cas9 to edit the grain size gene GW8 in the maintainer line WaitaiB (WTB) and restorer line Guanghui998 (GH998). The new slender sterile line WTEA (gw8) was obtained in the BC2F1 generation by transferring the grain mutation of the maintainer plant to the corresponding sterile line WantaiA (WTA, GW8) in the T1 generation. Two slender restorer lines, GH998E1 (gw8(II)) and GH998E2 (gw8(I)), were obtained in T1 generation. In the early stage, new sterile and restorer lines in grain mutations were created by targeted editing of GS3, TGW3, and GW8 genes. These parental lines were mated to detect the impact of grain-type mutations on hybrid rice yield and quality. Mutations in gs3, gw8, and tgw3 had a minimal impact on agronomic traits except the grain size and thousand-grain weight. The decrease in grain width in the combination mainly came from gw8/gw8, gs3/gs3 increased the grain length, gs3/gs3-gw8/gw8 had a more significant effect on the grain length, and gs3/gs3-gw8/gw8(I) contributed more to grain length than gs3/gs3-gw8/gw8(II). The heterozygous TGW3/tgw3 may not significantly increase grain length. Electron microscopy revealed that the low-chalky slender-grain variety had a cylindrical grain shape, a uniform distribution of endosperm cells, and tightly arranged starch grains. Quantitative fluorescence analysis of endospermdevelopment-related genes showed that the combination of slender grain hybrid rice caused by gs3 and gw8 mutations promoted endosperm development and improved appearance quality. An appropriate grain size mutation resulted in hybrid rice varieties with high yield and quality.

Molecular basis of genetic improvement for key rice quality traits in Southern China.

Grain qualities including milling quality, appearance quality, eating and cooking quality, and nutritional quality are important indicators in rice breeding. Significant achievements in genetic improvement of rice quality have been made. In this study, we analyzed the variation patterns of 16 traits in 1570 rice varieties and found significant improvements in appearance quality and eating and cooking quality, particularly in hybrid rice. Through genome-wide association study and allelic functional nucleotide polymorphisms analysis of quality trait genes, we found that ALK, FGR1, FLO7, GL7/GW7, GLW7, GS2, GS3, ONAC129, OsGRF8, POW1, WCR1, and Wx were associated with the genetic improvement of rice quality traits in Southern China. Allelic functional nucleotide polymorphisms analysis of 13 important rice quality genes, including fragrance gene fgr, were performed using the polymerase chain reaction amplification refractory mutation system technology. The results showed that Gui516, Gui569, Gui721, Ryousi, Rsimiao, Rbasi, and Yuehui9802 possessed multiple superior alleles. This study elucidates the phenotypic changes and molecular basis of key quality traits of varieties in Southern China. The findings will provide guidance for genetic improvement of rice quality and the development of new varieties.

Editing of rice (Oryza sativa L.) OsMKK3 gene using CRISPR/Cas9 decreases grain length by modulating the expression of photosystem components.

Grain size is one of the most important agronomic traits for grain yield determination in rice. To better understand the proteins that are regulated by the grain size regulatory gene OsMKK3, this gene was knocked out using the CRISPR/Cas9 system, and tandem mass tag (TMT) labeling combined with liquid chromatograph-tandem mass spectrometry analysis was performed to study the regulation of proteins in the panicle. Quantitative proteomic screening revealed a total of 106 differentially expressed proteins (DEPs) via comparison of the OsMKK3 mutant line to the wild-type YexiangB, including 15 and 91 up-regulated and down-regulated DEPs, respectively. Pathway analysis revealed that DEPs were enriched in metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and photosynthesis. Strong interactions were detected among seven down-regulated proteins related to photosystem components in the protein-protein interaction network, and photosynthetic rate was decreased in mutant plants. The results of the liquid chromatography-parallel reaction monitoring/mass spectromery analysis and western blot analysis were consistent with the results of the proteomic analysis, and the results of the quantitative reverse transcription polymerase chain reaction analysis revealed that the expression levels of most candidate genes were consistent with protein levels. Overall, OsMKK3 controls grain size by regulating the protein content in cells. Our findings provide new candidate genes that will aid the study of grain size regulatory mechanisms associated with the mitogen-activated protein kinase (MAPK) signaling pathway.

Utilization of natural alleles for heat adaptability QTLs at the flowering stage in rice.

BACKGROUND: Heat stress threatens rice yield and quality at flowering stage. In this study, average relative seed setting rate under heat stress (RHSR) and genotypes of 284 varieties were used for a genome-wide association study. RESULTS: We identified eight and six QTLs distributed on chromosomes 1, 3, 4, 5, 7 and 12 in the full population and indica, respectively. qHTT4.2 was detected in both the full population and indica as an overlapping QTL. RHSR was positively correlated with the accumulation of heat-tolerant superior alleles (SA), and indica accession contained at least two heat-tolerant SA with average RHSR greater than 43%, meeting the needs of stable production and heat-tolerant QTLs were offer yield basic for chalkiness degree, amylose content, gel consistency and gelatinization temperature. Chalkiness degree, amylose content, and gelatinization temperature under heat stress increased with accumulation of heat-tolerant SA. Gel consistency under heat stress decreased with polymerization of heat-tolerant SA. The study revealed qHTT4.2 as a stable heat-tolerant QTL that can be used for breeding that was detected in the full population and indica. And the grain quality of qHTT4.2-haplotype1 (Hap1) with chalk5, wx, and alk was better than that of qHTT4.2-Hap1 with CHALK5, WX, and ALK. Twelve putative candidate genes were identified for qHTT4.2 that enhance RHSR based on gene expression data and these genes were validated in two groups. Candidate genes LOC_Os04g52830 and LOC_Os04g52870 were induced by high temperature. CONCLUSIONS: Our findings identify strong heat-tolerant cultivars and heat-tolerant QTLs with great potential value to improve rice tolerance to heat stress, and suggest a strategy for the breeding of yield-balance-quality heat-tolerant crop varieties.

The genetic editing of GS3 via CRISPR/Cas9 accelerates the breeding of three-line hybrid rice with superior yield and grain quality.

Grain size is one of the major traits that determine rice grain yield and quality. The GS3 gene is the first major quantitative trait locus (QTL) that was identified in regulating rice grain length and weight. It was reported that the gs3 allele with a mutation in the organ size regulation (OSR) domain of the GS3 protein produced longer grains. In this study, we used the CRISPR/Cas9 gene editing technology to introduce an edited gs3 allele into our indica maintainer line, Mei1B, to enhance its grain yield and quality. Through molecular analysis and sequencing, a homologous edited-gs3 mutant line without any transgene was obtained in the T1 generation and was named Mei2B. A superior male sterile line Mei2A was generated by backcrossing the cytoplasmic male sterile (CMS) line Mei1A with Mei2B. Mei2B had a higher grain quality and yield compared to its wild-type Mei1B. Its grain length increased by 7.9%, its length/width ratio increased from 3.89 to 4.19, TGW increased by 6.7%, and grain yield per plant increased by 14.9%. In addition, genetic improvement of other quality traits including brown rice length (6.83 mm), brown rice grain length/width ratio (3.61), matched the appearance standards set for traditional Simiao (silk seedling) type cultivars. Two restorer lines were outcrossed to both Mei1A and Mei2A to produce hybrid rice. Compared to two hybrids of Mei1A, the hybrids of Mei2A had longer grains, higher length/width ratio, TGW, and yield per plant. In addition, the hybrids of Mei2A showed a better grain appearance including better translucency, a lower chalky rice rate, and degree of chalkiness than the hybrids of Mei1A. These results demonstrated that the introduction of an elite gs3 allele into Mei1A via CRISPR/Cas9 gene editing technology led to significant genetic improvement of the rice grain. The resultant CMS line Mei2A(gs3) displayed much higher grain quality and yield than the original Mei1A. Therefore, our study demonstrated that the targeted genetic improvement via gene editing technology can enhance rice breeding, especially the breeding of three-line hybrid rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01290-z.

ITRAQ-based quantitative proteomic analysis of japonica rice seedling during cold stress.

Low temperature is one of the important environmental factors that affect rice growth and yield. To better understand the japonica rice responses to cold stress, isobaric tags for a relative and absolute quantification (iTRAQ) labeling-based quantitative proteomics approach was used to detected changes in protein levels. Two-week-old seedlings of the cold tolerant rice variety Kongyu131 were treated at 8°C for 24, 48 and 72 h, then the total proteins were extracted from tissues and used for quantitative proteomics analysis. A total of 5082 proteins were detected for quantitative analysis, of which 289 proteins were significantly regulated, consisting of 169 uniquely up-regulated proteins and 125 uniquely down-regulated proteins in cold stress groups relative to the control group. Functional analysis revealed that most of the regulated proteins are involved in photosynthesis, metabolic pathway, biosynthesis of secondary metabolites and carbon metabolism. Western blot analysis showed that protein regulation was consistent with the iTRAQ data. The corresponding genes of 25 regulated proteins were used for quantitative real time PCR analysis, and the results showed that the mRNA level was not always parallel to the corresponding protein level. The importance of our study is that it provides new insights into cold stress responses in rice with respect to proteomics and provides candidate genes for cold-tolerance rice breeding.

Quantitative and functional posttranslational modification proteomics reveals that TREPH1 plays a role in plant touch-delayed bolting.

Environmental mechanical forces, such as wind and touch, trigger gene-expression regulation and developmental changes, called "thigmomorphogenesis," in plants, demonstrating the ability of plants to perceive such stimuli. In Arabidopsis, a major thigmomorphogenetic response is delayed bolting, i.e., emergence of the flowering stem. The signaling components responsible for mechanotransduction of the touch response are largely unknown. Here, we performed a high-throughput SILIA (stable isotope labeling in Arabidopsis)-based quantitative phosphoproteomics analysis to profile changes in protein phosphorylation resulting from 40 seconds of force stimulation in Arabidopsis thaliana Of the 24 touch-responsive phosphopeptides identified, many were derived from kinases, phosphatases, cytoskeleton proteins, membrane proteins, and ion transporters. In addition, the previously uncharacterized protein TOUCH-REGULATED PHOSPHOPROTEIN1 (TREPH1) became rapidly phosphorylated in touch-stimulated plants, as confirmed by immunoblots. TREPH1 fractionates as a soluble protein and is shown to be required for the touch-induced delay of bolting and gene-expression changes. Furthermore, a nonphosphorylatable site-specific isoform of TREPH1 (S625A) failed to restore touch-induced flowering delay of treph1-1, indicating the necessity of S625 for TREPH1 function and providing evidence consistent with the possible functional relevance of the touch-regulated TREPH1 phosphorylation. Taken together, these findings identify a phosphoprotein player in Arabidopsis thigmomorphogenesis regulation and provide evidence that TREPH1 and its touch-induced phosphorylation may play a role in touch-induced bolting delay, a major component of thigmomorphogenesis.

Transcriptomic profiling of germinating seeds under cold stress and characterization of the cold-tolerant gene LTG5 in rice.

BACKGROUND: Low temperature is a limiting factor of rice productivity and geographical distribution. Wild rice (Oryza rufipogon Griff.) is an important germplasm resource for rice improvement. It has superior tolerance to many abiotic stresses, including cold stress, but little is known about the mechanism underlying its resistance to cold. RESULTS: This study elucidated the molecular genetic mechanisms of wild rice in tolerating low temperature. Comprehensive transcriptome profiles of two rice genotypes (cold-sensitive ce 253 and cold-tolerant Y12-4) at the germinating stage under cold stress were comparatively analyzed. A total of 42.44-68.71 million readings were obtained, resulting in the alignment of 29,128 and 30,131 genes in genotypes 253 and Y12-4, respectively. Many common and differentially expressed genes (DEGs) were analyzed in the cold-sensitive and cold-tolerant genotypes. Results showed more upregulated DEGs in the cold-tolerant genotype than in the cold-sensitive genotype at four stages under cold stress. Gene ontology enrichment analyses based on cellular process, metabolic process, response stimulus, membrane part, and catalytic activity indicated more upregulated genes than downregulated ones in the cold-tolerant genotype than in the cold-sensitive genotype. Quantitative real-time polymerase chain reaction was performed on seven randomly selected DEGs to confirm the RNA Sequencing (RNA-seq) data. These genes showed similar expression patterns corresponding with the RNA-Seq method. Weighted gene co-expression network analysis (WGCNA) revealed Y12-4 showed more positive genes than 253 under cold stress. We also explored the cold tolerance gene LTG5 (Low Temperature Growth 5) encoding a UDP-glucosyltransferase. The overexpression of the LTG5 gene conferred cold tolerance to indica rice. CONCLUSION: Gene resources related to cold stress from wild rice can be valuable for improving the cold tolerance of crops.

Development of molecular marker and introgression of Bph3 into elite rice cultivars by marker-assisted selection.

The brown planthopper (BPH) is a serious insect pest of rice and a substantial threat to rice production. Identification of new BPH resistance genes and their transfer into modern rice cultivars are effective breeding approaches to reduce the damage caused by BPH. In this study, we mapped a BPH resistance gene to a 50-kb genomic interval between two InDel markers 4M03980 and 4M04041 on the short arm of chromosome 4 in indica rice cultivar BP60, where the BPH resistance gene was mapped in Rathu Heenati by Liu et al. (2015) and named "Bph3". This region contains two annotated genes Os04g0201900 and Os04g0202300, which encode lectin receptor kinases responsible for BPH resistance. We also developed a molecular marker "MM28T" for Bph3, and introgression Bph3 into susceptible rice restorer lines Guihui582 and Gui7571 by the marker-assisted selection (MAS) approach. The BPH resistance level is significantly enhanced in the Bph3-introgression lines, the resistance scores decrease from 8.2 to 3.6 for Guihui582 and decrease from 8.7 to around 3.8 for Gui7571. Therefore, developing molecular markers for the BPH resistance gene Bph3 and using them for molecular breeding will facilitate the creation of BPH-resistance rice cultivars to reduce damage caused by BPH.

Mapping and Identifying a Candidate Gene Plr4, a Recessive Gene Regulating Purple Leaf in Rice, by Using Bulked Segregant and Transcriptome Analysis with Next-Generation Sequencing.

The anthocyanin biosynthesis of rice is a major concern due to the potential nutritional value. Purple appears in various organs and tissues of rice such as pericarp, flower organs, leaves, leaf sheaths, internodes, ligules, apex, and stigma. At present, there are many studies on the color of rice pericarp, but the gene and mechanism of other organs such as leaves are still unclear, and the gene regulatory network of specific organ coloring has not been systematically understood. In this study, genetic analysis demonstrated that the purple leaf traits of rice were regulated by a recessive gene. The green leaf cultivar Y58S and purple leaf cultivar XianHongB were used to construct the mapping population. A set of near isogenicline (NIL) (BC3F1) was bred via crossing and back-crossing. The generations of BC3F2 appeared to separate four phenotypes, pl1, pl2, pl3, and pl4, due to the occurrence of a purple color in different organs. We constructed three bulked segregant analysis (BSA) pools (pl1-pl2, pl1-pl3, and pl1-pl4) by using the separated generations of BC3F5 and mapped the purple leaf gene plr4 to the vicinity of 27.9-31.1 Mb on chromosome 4. Subsequently, transcriptome sequencing (RNA-Seq) for pl3 and pl2 was used to analyze the differentially expressed genes in the localization interval, where 12 unigenes exhibited differential expression in which two genes (Os04g0577800, Os04g0616400) were downregulated. The two downregulated genes (Os04g0577800 and Os04g0616400) are possible candidate genes because of the recessive genetic characteristics of the purple leaf genes. These results will facilitate the cloning of plr4 and illustrate the molecular mechanisms of the anthocyanin synthesis pathway.

The Landscape of Presence/Absence Variations during the Improvement of Rice.

Rice is one of the most important staple crops in the world; therefore, the improvement of rice holds great significance for enhancing agricultural production and addressing food security challenges. Although there have been numerous studies on the role of single-nucleotide polymorphisms (SNPs) in rice improvement with the development of next-generation sequencing technologies, research on the role of presence/absence variations (PAVs) in the improvement of rice is limited. In particular, there is a scarcity of studies exploring the traits and genes that may be affected by PAVs in rice. Here, we extracted PAVs utilizing resequencing data from 148 improved rice varieties distributed in Asia. We detected a total of 33,220 PAVs and found that the number of variations decreased gradually as the length of the PAVs increased. The number of PAVs was the highest on chromosome 1. Furthermore, we identified a 6 Mb hotspot region on chromosome 11 containing 1091 PAVs in which there were 29 genes related to defense responses. By conducting a genome-wide association study (GWAS) using PAV variation data and phenotypic data for five traits (flowering time, plant height, flag leaf length, flag leaf width, and panicle number) across all materials, we identified 186 significantly associated PAVs involving 20 cloned genes. A haplotype analysis and expression analysis of candidate genes revealed that important genes might be affected by PAVs, such as the flowering time gene OsSFL1 and the flag leaf width gene NAL1. Our work investigated the pattern in PAVs and explored important PAV key functional genes associated with agronomic traits. Consequently, these results provide potential and exploitable genetic resources for rice breeding.

Genetic Effects of Grain Quality Enhancement in Indica Hybrid Rice: Insights for Molecular Design Breeding.

Improving rice quality remains a crucial breeding objective, second only to enhancing yield, yet progress in quality improvement lags behind yield. The high temperature and ripening conditions in Southern China often result in poor rice quality, impacting hybrid rice production and utilization. Therefore, to address this challenge, analyzing the molecular basis of high-quality traits is essential for molecular design breeding of high-quality hybrid rice varieties. In this study, we investigated the molecular basis of grain shape, amylose content, gel consistency, gelatinization temperature, and aroma, which influence rice quality. We discovered that quality related alleles gs3, GW7TFA, gw8, chalk5, Wxb, ALKTT, and fgr can enhance rice quality when applied in breeding programs. Polymerization of gs3, GW7TFA, gw8, and chalk5 genes improves rice appearance quality. The gs3 and GW7TFA allele polymerization increasing the grain's length-width ratio, adding the aggregation of gw8 allele can further reducing grain width. The chalk5 gene regulates low chalkiness, but low correlation to chalkiness was exhibited with grain widths below 2.0 mm, with minimal differences between Chalk5 and chalk5 alleles. Enhancing rice cooking and eating quality is achieved through Wxb and ALKTT gene polymerization, while introducing the fgr(E7) gene significantly improved rice aroma. Using molecular marker-assisted technology, we aggregated these genes to develop a batch of indica hybrid rice parents with improved rice quality are obtained. Cross-combining these enhanced parents can generate new, high-quality hybrid rice varieties suitable for cultivation in Southern China. Therefore, our findings contribute to a molecular breeding model for grain quality improvement in high-quality indica hybrid rice. This study, along with others, highlights the potential of molecular design breeding for enhancing complex traits, particularly rice grain quality.

TMT-based quantitative proteomic analysis of indica rice cultivars reveals that novel components of the signaling pathways might play a role in grain length regulation.

Grain length is one of the most important rice grain appearance components. To better understand the protein regulated by grain length in indica rice, the tandem mass tag (TMT) labeling combined with LC-MS/MS analysis was used for quantitative identification of differentially regulated proteins by comparing six long-grain cultivars (MeiB, LongfengB, YexiangB, FengtianB, WantaiB, and DingxiangB) to the short-grain cultivar BoB, respectively. A total of 6622 proteins were detected for quantitative analysis by comparing protein content of six long-grain cultivars to the short-grain cultivar, and 715 proteins were significantly regulated, consisting of 336 uniquely over-accumulated proteins and 355 uniquely down-accumulated proteins. KEGG pathway analysis revealed that most of accumulated proteins are involved in metabolic pathways, biosynthesis of secondary metabolites and phenylpropanoid biosynthesis. Four down-accumulated proteins maybe involved in the signaling pathways for grain length regulation. LC-PRM/MS quantitative analysis was used to analyze 10 differentially expressed proteins. The results were almost consistent with the TMT quantitative analysis. qRT-PCR analysis results showed that the transcription level was not always parallel to the protein content. This study identified many novel grain length accumulated proteins through the quantitative proteomics approach, providing candidate genes for further study of grain size regulatory mechanisms. SIGNIFICANCE: Rice grain length is one of the most important characteristics influencing appearance and yield. Six long-grain cultivars (MeiB, LongfengB, YexiangB, FengtianB, WantaiB, and DingxiangB obtained in Guangxi province of China from the 2000s to 2020s) and one short-grain cultivar (BoB obtained in Guangxi province of China in 1980s) were used for comparative analyses. Totally, 715 differentially expressed proteins (DEPs) were identified using TMT-base proteomic analysis. The numbers of DEPs increased as the grain length increased. 4 DEPs may be related to rice's signaling pathways for grain size regulation. A total of 85 DEPs regulated in at least four long-grain cultivars compared with the short-grain cultivar BoB, and 7 proteins were over-accumulated, and 3 proteins were down-accumulated in six long-grain cultivars. These findings provide valuable information to better understand the mechanisms of protein regulation by grain length in rice.

QTL mapping and identification of candidate genes for cold tolerance at the germination stage in wild rice.

BACKGROUND: Cold damage stress significantly affects rice growth (germination and seedling) and causes serious losses in yield in temperate and high-altitude areas around the globe. OBJECTIVE: This study aimed to explore the cold tolerance (CT) locus of rice and create new cold-tolerant germplasm. We constructed a chromosome segment substitution line (CSSL) with strong CT and fine mapped quantitative trait loci (QTLs) associated with CT by performing the whole-genome resequencing of CSSL with phenotypes under cold treatment. METHODS: A chromosome CSSL, including 271 lines from a cross between the cold-tolerant wild rice Y11 (Oryza rufipogon Griff.) and the cold-sensitive rice variety GH998, was developed to map QTLs conferring CT at the germination stage. The whole-genome resequencing was performed on CSSL for mapping QTLs of associated with CT at the germination stage. RESULTS: A high-density linkage map of the CSSLs was developed using the whole-genome resequencing of 1484 bins. The QTL analysis using 615,466 single-nucleotide polymorphisms (SNPs) led to the identification of 2 QTLs related to germination rate at low-temperature on chromosome 8 (qCTG-8) and chromosome 11 (qCTG-11). The qCTG-8 and qCTG-11 explained 14.55% and 14.31% of the total phenotypic variation, respectively. We narrowed down qCTG-8 and qCTG-11 to 195.5 and 78.83-kb regions, respectively. The expression patterns of important candidate genes in different tissues, and of RNA-sequencing (RNA-seq) in CSSLs, were identified based on gene sequences in qCTG-8 and qCTG-11 cold-induced expression analysis. LOC_Os08g01120 and LOC_Os08g01390 were identified as candidate genes in qCTG-8, and LOC_Os11g32880 was identified as a candidate gene in qCTG-11. CONCLUSIONS: This study demonstrated a general method that could be used to identify useful loci and genes in wild rice and aid in the future cloning of candidate genes of qCTG-8 and qCTG-11. The CSSLs with strong CT were supported for breeding cold-tolerant rice varieties.

Systematic Analysis of NB-ARC Gene Family in Rice and Functional Characterization of GNP12.

The NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) gene family plays a critical role in plant development. However, our understanding of the mechanisms of how NB-ARC genes regulate plant development in the plant panicle is still limited. Here, we subjected 258 NB-ARC genes in rice to genome-wide analysis to characterize their structure, function, and expression patterns. The NB-ARC genes were classified into three major groups, and group II included nine subgroups. Evolutionary analysis of NB-ARC genes in a dicotyledon plant (Arabidopsis thaliana) and two monocotyledonous plants (Oryza sativa L. and Triticum aestivum) indicated that homologous genome segments were conserved in monocotyledons and subjected to weak positive selective pressure during evolution. Dispersed and proximal replication events were detected. Expression analysis showed expression of most NB-ARC genes in roots, panicles, and leaves, and regulation at the panicle development stage in rice Ce253. The GNP12 gene encodes RGH1A protein, which regulates rice yield according to panicle length, grain number of panicle, and grain length, with eight major haplotypes. Most members of NB-ARC protein family are predicted to contain P-loop conserved domains and localize on the membrane. The results of this study will provide insight into the characteristics and evolution of NB-ARC family and suggest that GNP12 positively regulates panicle development.

The panorama of physiological responses and gene expression of whole plant of maize inbred line YQ7-96 at the three-leaf stage under water deficit and re-watering.

Changes in water potential, growth elongation, photosynthesis of three-leaf-old seedlings of maize inbred line YQ7-96 under water deficit (WD) for 0.5, 1 and 2 h and re-watering (RW) for 24 h were characterized. Gene expression was analyzed using cDNA microarray covering 11,855 maize unigenes. As for whole maize plant, the expression of WD-regulated genes was characterized by up-regulation. The expression of WD-regulated genes was categorized into eight different patterns, respectively, in leaves and roots. Newly found and WD-affected cellular processes were metabolic process, amino acid and derivative metabolic process and cell death. A great number of the analyzed genes were found to be regulated specifically by RW and commonly by both WD and RW, respectively, in leaves. It is therefore concluded that (1) whole maize plant tolerance to WD, as well as growth recovery from WD, depends at least in part on transcriptional coordination between leaves and roots; (2) WD exerts effects on the maize, especially on basal metabolism; (3) WD could probably affect CO(2) uptake and partitioning, and transport of fixed carbons; (4) WD could likely influence nuclear activity and genome stability; and (5) maize growth recovery from WD is likely involved in some specific signaling pathways related to RW-specific responsive genes.

Comparative profiles of gene expression in leaves and roots of maize seedlings under conditions of salt stress and the removal of salt stress.

We studied the transcriptional profiles of leaves and roots of three-leaf stage seedlings of the maize inbred line YQ7-96 under conditions of salt stress (100 mM NaCl) and removal of salt stress (RSS). A total of 296 genes were regulated specifically by the stress, of which 206 were specific to leaves and 90 were specific to roots. Stress-regulated genes were classified into eight and seven expression patterns for leaves and roots, respectively. There were 60 genes which were regulated specifically by RSS, 27 of which were specific to leaves and 33 specific to roots. No genes were found to be co-regulated in tissues and to be regulated commonly by the stress and RSS. It can be concluded that (i) at the early stage of the stress, transcriptional responses are directed at water deficit in maize leaves but at both water deficit and Na+ accumulation in roots; (ii) at the later stage, the responses in leaves and roots result from dual effects of both water deficit and Na+ accumulation; (iii) the polyamine metabolic pathway is an important linker for the co-ordination between leaves and roots to accomplish the tolerance of the whole maize plant to the stress; (iv) the stress can lead to genomic restructuring and nuclear transport in maize; (v) maize leaves are distinct from roots in terms of molecular mechanisms for responses to and growth recovery from the stress; and (vi) mechanisms for the maize responses to the stress differ from those for their growth recovery during RSS.

Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-Terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis.

Ethylene participates in the regulation of numerous cellular events and biological processes, including water loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a (15)N stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eil1-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene-regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-0 and ein3eil1 genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up-regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs.