Genome-wide identification and comprehensive analysis of tubby-like protein gene family in multiple crops.

Introduction: The highly conserved tubby-like proteins (TLPs) play key roles in animal neuronal development and plant growth. The abiotic stress tolerance function of TLPs has been widely explored in plants, however, little is known about comparative studies of TLPs within crops. Methods: Bioinformatic identification, phylogenetic analysis, Cis-element analysis, expression analysis, Cis-element analysis, expression analysis and so on were explored to analysis the TLP gene family of multiple crops. Results: In this study, a comprehensive analysis of TLP genes were carried out in seven crops to explore whether similar function of TLPs in rice could be achieved in other crops. We identified 20, 9, 14, 11, 12, 35, 14 and 13 TLP genes in Glycine max, Hordeum vulgare, Sorghum bicolor, Arabidopsis thaliana, Oryza sativa Japonica, Triticum aestivum, Setaria italic and Zea mays, respectively. All of them were divided into two groups and ten orthogroups (Ors) based on amino acids. A majority of TLP genes had two domains, tubby-like domain and F-box domain, while members of Or5 only had tubby-like domain. In addition, Or5 had more exons and shorter DNA sequences, showing that characteristics of different Ors reflected the differentiated function and feature of TLP genes in evolutionary process, and Or5 was the most different from the other Ors. Besides, we recognized 25 cis-elements in the promoter of TLP genes and explored multiple new regulation pathway of TLPs including light and hormone response. The bioinformatic and transcriptomic analysis implied the stresses induced expression and possible functional redundancy of TLP genes. We detected the expression level of 6 OsTLP genes at 1 to 6 days after seed germination in rice, and the most obvious changes in these days were appeared in OsTLP10 and OsTLP12. Discussion: Combined yeast two-hybrid system and pull down assay, we suggested that the TLP genes of Or1 may have similar function during seed germination in different species. In general, the results of comprehensive analysis of TLP gene family in multiple species provide valuable evolutionary and functional information of TLP gene family which are useful for further application and study of TLP genes.

Genic male sterility increases rice drought tolerance.

Plant fertility and resistance to stress environments are antagonistic to each other. At booting stage, fertility is often sacrificed for survive in rice under abiotic stress. However, the relationship between fertility and resistance at molecular level remains elusive. Here, we identified a transcription factor, OsAlfin like 5, which regulates the OsTMS5 and links both the drought stress response and thermosensitive genic male sterility. The OsAL5 overexpression plants (OE-OsAL5) became sensitive to temperature owning to the OsTMS5 that the OE-OsAL5 plants were fertile under low temperature (23 °C) and sterile under high temperature (28 °C). Significantly, the survival rate of OE-OsAL5 lines was higher than that of the wide-type (WT) under drought stress. Further experiments confirmed that the OsAL5 regulated both of the OsTMS5 and the down-stream drought-related genes by binding to the 'GTGGAG' element in vivo, revealing that the OsAL5 participated both in the drought stress response and thermosensitive genic male sterility in rice. These findings open up the possibility of breeding elite TGMS lines with strong drought tolerance by manipulating the expression of OsAL5.

The transcription factor OsbHLH138 regulates thermosensitive genic male sterility in rice via activation of TMS5.

Thermosensitive genic male sterile (TGMS) lines favored heterosis exploitation in two-line hybrid rice. TMS5, a member of RNase Z cleavages the UbL40 mRNAs, plays an important role in two-line hybrid rice. Here, we identified a new TGMS mutant 93-11s, which lost two amino acids in the first exon of TMS5 gene and caused thermosensitive genic male sterility in rice. The tms5-2 cannot process mRNAs of the ubiquitin fusion ribosomal protein L40 (UbL40) and hence cause the mRNAs accumulation in restrictive temperature. Further, we identified a nucleus-localized bHLH transcription factor OsbHLH138, which can form the basic helix-loop-helix structure and bind the core region of tms5-2 promoter sequences by bHLH domain, and activate expression of tms5-2 by the acidic amino acid-rich domain. These results indicate a novel mechanism for the tms5-2 regulating thermosensitive male sterility of rice. By altering expression of OsbHLH138, we can regulate the expression level of TMS5 and the accumulation of UbL40 mRNAs to command the male fertility in different temperatures. The identification of OsbHLH138 provides breeders a new choice for development of TGMS rice lines, which will favor the sustainable development of two-line hybrid rice.

Rational Improvement of Rice Yield and Cold Tolerance by Editing the Three Genes OsPIN5b, GS3, and OsMYB30 With the CRISPR-Cas9 System.

Significant increases in rice yield and stress resistance are constant demands for breeders. However, high yield and high stress resistance are often antagonistic to each other. Here, we report several new rice mutants with high yield and excellent cold tolerance that were generated by simultaneously editing three genes, OsPIN5b (a panicle length gene), GS3 (a grain size gene) and OsMYB30 (a cold tolerance gene) with the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) system. We edited two target sites of each gene with high efficiency: 53% for OsPIN5b-site1, 42% for OsPIN5b-site2, 66% for GS3-site1, 63% for GS3-site2, 63% for OsMYB30-site1, and 58% for OsMYB30-site2. Consequently, the ospin5b mutants, the gs3 mutants, and the osmyb30 mutants exhibited increased panicle length, enlarged grain size and increased cold tolerance, respectively. Then nine transgenic lines of the ospin5b/gs3, six lines of ospin5b/osmyb30 and six lines of gs3/osmyb30 were also acquired, and their yield related traits and cold tolerance corresponded to the genes being edited. Additionally, we obtained eight ospin5b/gs3/osmyb30 triple mutants by editing all three genes simultaneously. Aside from the ospin5b/gs3/osmyb30-4 and ospin5b/gs3/osmyb30-25 mutants, the remaining six mutants had off-target events at the putative off-target site of OsMYB30-site1. The results also showed that the T2 generations of these two mutants exhibited higher yield and better cold tolerance compared with the wild type. Together, these results demonstrated that new and excellent rice varieties with improved yield and abiotic stress resistance can be generated through gene editing techniques and may be applied to rice breeding. Furthermore, our study proved that the comprehensive agronomic traits of rice can be improved with the CRISPR-Cas9 system.