Yongbiao Xue

Sequence and analysis of rice chromosome 4

Rice is the principal food for over half of the population of the world. With its genome size of 430 megabase pairs (Mb), the cultivated rice species Oryza sativa is a model plant for genome research. Here we report the sequence analysis of chromosome 4 of O. sativa, one of the first two rice chromosomes to be sequenced completely. The finished sequence spans 34.6 Mb and represents 97.3% of the chromosome. In addition, we report the longest known sequence for a plant centromere, a completely sequenced contig of 1.16 Mb corresponding to the centromeric region of chromosome 4. We predict 4,658 protein coding genes and 70 transfer RNA genes. A total of 1,681 predicted genes match available unique rice expressed sequence tags. Transposable elements have a pronounced bias towards the euchromatic regions, indicating a close correlation of their distributions to genes along the chromosome. Comparative genome analysis between cultivated rice subspecies shows that there is an overall syntenic relationship between the chromosomes and divergence at the level of single-nucleotide polymorphisms and insertions and deletions. By contrast, there is little conservation in gene order between rice and Arabidopsis.

Overexpression of an R1R2R3 MYB Gene, OsMYB3R-2, Increases Tolerance to Freezing, Drought, and Salt Stress in Transgenic Arabidopsis

We used a cDNA microarray approach to monitor the expression profile of rice (Oryza sativa) under cold stress and identified 328 cold-regulated genes. Thirteen such genes encoding MYB, homeodomain, and zinc finger proteins with unknown functions showed a significant change in expression under 72-h cold stress. Among them, OsMYB3R-2 was selected for further study. Unlike most plant R2R3 MYB transcription factors, OsMYB3R-2 has three imperfect repeats in the DNA-binding domain, the same as in animal c-MYB proteins. Expression of OsMYB3R-2 was induced by cold, drought, and salt stress. The Arabidopsis (Arabidopsis thaliana) transgenic plants overexpressing OsMYB3R-2 showed increased tolerance to cold, drought, and salt stress, and the seed germination of transgenic plants was more tolerant to abscisic acid or NaCl than that of wild type. The expression of some clod-related genes, such as dehydration-responsive element-binding protein 2A, COR15a, and RCI2A, was increased to a higher level in OsMYB3R-2-overexpressing plants than in wild type. These results suggest that OsMYB3R-2 acts as a master switch in stress tolerance.

Genomic analyses provide insights into the history of tomato breeding

The histories of crop domestication and breeding are recorded in genomes. Although tomato is a model species for plant biology and breeding, the nature of human selection that altered its genome remains largely unknown. Here we report a comprehensive analysis of tomato evolution based on the genome sequences of 360 accessions. We provide evidence that domestication and improvement focused on two independent sets of quantitative trait loci (QTLs), resulting in modern tomato fruit ∼100 times larger than its ancestor. Furthermore, we discovered a major genomic signature for modern processing tomatoes, identified the causative variants that confer pink fruit color and precisely visualized the linkage drag associated with wild introgressions. This study outlines the accomplishments as well as the costs of historical selection and provides molecular insights toward further improvement.

A triallelic system of S5 is a major regulator of the reproductive barrier and compatibility of indica-japonica hybrids in rice

Hybrid sterility is a major form of postzygotic reproductive isolation. Although reproductive isolation has been a key issue in evolutionary biology for many decades in a wide range of organisms, only very recently a few genes for reproductive isolation were identified. The Asian cultivated rice (Oryza sativa L.) is divided into two subspecies, indica and japonica. Hybrids between indica and japonica varieties are usually highly sterile. A special group of rice germplasm, referred to as wide-compatibility varieties, is able to produce highly fertile hybrids when crossed to both indica and japonica. In this study, we cloned S5, a major locus for indica–japonica hybrid sterility and wide compatibility, using a map-based cloning approach. We show that S5 encodes an aspartic protease conditioning embryo-sac fertility. The indica (S5-i) and japonica (S5-j) alleles differ by two nucleotides. The wide compatibility gene (S5-n) has a large deletion in the N terminus of the predicted S5 protein, causing subcellular mislocalization of the protein, and thus is presumably nonfunctional. This triallelic system has a profound implication in the evolution and artificial breeding of cultivated rice. Genetic differentiation between indica and japonica would have been enforced because of the reproductive barrier caused by S5-i and S5-j, and species coherence would have been maintained by gene flow enabled by the wide compatibility gene.

The F-Box Protein AhSLF-S2 Physically Interacts with S-RNases That May Be Inhibited by the Ubiquitin/26S Proteasome Pathway of Protein Degradation during Compatible Pollination in Antirrhinum

Self-incompatibility S-locus–encoded F-box (SLF) proteins have been identified in Antirrhinum and several Prunus species. Although they appear to play an important role in self-incompatible reaction, functional evidence is lacking. Here, we provide several lines of evidence directly implicating a role of AhSLF-S2 in self-incompatibility in Antirrhinum. First, a nonallelic physical interaction between AhSLF-S2 and S-RNases was demonstrated by both coimmunoprecipitation and yeast two-hybrid assays. Second, AhSLF-S2 interacts with ASK1- and CULLIN1-like proteins in Antirrhinum, and together, they likely form an Skp1/Cullin or CDC53/F-box (SCF) complex. Third, compatible pollination was specifically blocked after the treatment of the proteasomal inhibitors MG115 and MG132, but they had little effect on incompatible pollination both in vitro and in vivo, indicating that the ubiquitin/26S proteasome activity is involved in compatible pollination. Fourth, the ubiquitination level of style proteins was increased substantially after compatible pollination compared with incompatible pollination, and coimmunoprecipitation revealed that S-RNases were ubiquitinated after incubating pollen proteins with compatible but not with incompatible style proteins, suggesting that non-self S-RNases are possibly degraded by the ubiquitin/26S proteasome pathway. Fifth, the S-RNase level appeared to be reduced after 36 h of compatible pollination. Taken together, these results show that AhSLF-S2 interacts with S-RNases likely through a proposed SCFAhSLF-S2 complex that targets S-RNase destruction during compatible rather than incompatible pollination, thus providing a biochemical basis for the inhibition of pollen tube growth as observed in self-incompatible response in Antirrhinum.

The F-Box Protein AhSLF-S2 Controls the Pollen Function of S-RNase–Based Self-Incompatibility

Recently, we have provided evidence that the polymorphic self-incompatibility (S) locus-encoded F-box (SLF) protein AhSLF-S2 plays a role in mediating a selective S-RNase destruction during the self-incompatible response in Antirrhinum hispanicum. To investigate its role further, we first transformed a transformation-competent artificial chromosome clone (TAC26) containing both AhSLF-S2 and AhS2-RNase into a self-incompatible (SI) line of Petunia hybrida. Molecular analyses showed that both genes are correctly expressed in pollen and pistil in four independent transgenic lines of petunia. Pollination tests indicated that all four lines became self-compatible because of the specific loss of the pollen function of SI. This alteration was transmitted stably into the T1 progeny. We then transformed AhSLF-S2 cDNA under the control of a tomato (Lycopersicon esculentum) pollen-specific promoter LAT52 into the self-incompatible petunia line. Molecular studies revealed that AhSLF-S2 is specifically expressed in pollen of five independent transgenic plants. Pollination tests showed that they also had lost the pollen function of SI. Importantly, expression of endogenous SLF or SLF-like genes was not altered in these transgenic plants. These results phenocopy a well-known phenomenon called competitive interaction whereby the presence of two different pollen S alleles within pollen leads to the breakdown of the pollen function of SI in several solanaceaous species. Furthermore, we demonstrated that AhSLF-S2 physically interacts with PhS3-RNase from the P. hybrida line used for transformation. Together with the recent demonstration of PiSLF as the pollen determinant in P. inflata, these results provide direct evidence that the polymorphic SLF including AhSLF-S2 controls the pollen function of S-RNase–based self-incompatibility.

A Novel Nuclear-Localized CCCH-Type Zinc Finger Protein, OsDOS, Is Involved in Delaying Leaf Senescence in Rice

Leaf senescence is a developmentally programmed degeneration process, which is fine tuned by a complex regulatory network for plant fitness. However, molecular regulation of leaf senescence is poorly understood, especially in rice (Oryza sativa), an important staple crop for more than half of the world population. Here, we report a novel nuclear-localized CCCH-type zinc finger protein, Oryza sativa delay of the onset of senescence (OsDOS), involved in delaying leaf senescence in rice. The expression of OsDOS was down-regulated during natural leaf senescence, panicle development, and pollination, although its transcripts were accumulated in various organs. RNAi knockdown of OsDOS caused an accelerated age-dependent leaf senescence, whereas its overexpression produced a marked delay of leaf senescence, suggesting that it acts as a negative regulator for leaf senescence. A genome-wide expression analysis further confirmed its negative regulation for leaf senescence and revealed that, in particular, the jasmonate (JA) pathway was found to be hyperactive in the OsDOS RNAi transgenic lines but impaired in the OsDOS overexpressing transgenic lines, indicating that this pathway is likely involved in the OsDOS-mediated delaying of leaf senescence. Furthermore, methyl JA treatments of both seeds and detached leaves from the RNAi and the overexpressing transgenic lines showed hyper- and hyporesponses, respectively, consistent with the negative regulation of the JA pathway by OsDOS. Together, these results indicate that OsDOS is a novel nuclear protein that delays leaf senescence likely, at least in part, by integrating developmental cues to the JA pathway.

Evolution in Action: Following Function in Duplicated Floral Homeotic Genes

Gene duplication plays a fundamental role in evolution by providing the genetic material from which novel functions can arise. Newly duplicated genes can be maintained by subfunctionalization (the duplicated genes perform different aspects of the original genes function) and/or neofunctionalization (one of the genes acquires a novel function). PLENA in Antirrhinum and AGAMOUS in Arabidopsis are the canonical C-function genes that are essential for the specification of reproductive organs. These functionally equivalent genes encode closely related homeotic MADS-box transcription factors. Using genome synteny, we confirm phylogenetic analyses showing that PLENA and AGAMOUS are nonorthologous genes derived from a duplication in a common ancestor. Their respective orthologs, SHATTERPROOF in Arabidopsis and FARINELLI in Antirrhinum, have undergone independent subfunctionalization via changes in regulation and protein function. Surprisingly, the functional divergence between PLENA and FARINELLI, is morphologically manifest in both transgenic Antirrhinum and Arabidopsis. This provides a clear illustration of a random evolutionary trajectory for gene functions after a duplication event. Different members of a duplicated gene pair have retained the primary homeotic functions in different lineages, illustrating the role of chance in evolution. The differential ability of the Antirrhinum genes to promote male or female development provides a striking example of subfunctionalization at the protein level.

The Tomato Sequencing Project, the First Cornerstone of the International Solanaceae Project (SOL)

The genome of tomato (Solanum lycopersicum) is being sequenced by an international consortium of 10 countries (Korea, China, the United Kingdom, India, The Netherlands, France, Japan, Spain, Italy and the United States) as part of a larger initiative called the ‘International Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation’. The goal of this grassroots initiative, launched in November 2003, is to establish a network of information, resources and scientists to ultimately tackle two of the most significant questions in plant biology and agriculture: (1) How can a common set of genes/proteins give rise to a wide range of morphologically and ecologically distinct organisms that occupy our planet? (2) How can a deeper understanding of the genetic basis of plant diversity be harnessed to better meet the needs of society in an environmentally friendly and sustainable manner? The Solanaceae and closely related species such as coffee, which are included in the scope of the SOL project, are ideally suited to address both of these questions. The first step of the SOL project is to use an ordered BAC approach to generate a high quality sequence for the euchromatic portions of the tomato as a reference for the Solanaceae. Due to the high level of macro and micro-synteny in the Solanaceae the BAC-by-BAC tomato sequence will form the framework for shotgun sequencing of other species. The starting point for sequencing the genome is BACs anchored to the genetic map by overgo hybridization and AFLP technology. The overgos are derived from approximately 1500 markers from the tomato high density F2-2000 genetic map (http://sgn.cornell.edu/). These seed BACs will be used as anchors from which to radiate the tiling path using BAC end sequence data. Annotation will be performed according to SOL project guidelines. All the information generated under the SOL umbrella will be made available in a comprehensive website. The information will be interlinked with the ultimate goal that the comparative biology of the Solanaceae—and beyond—achieves a context that will facilitate a systems biology approach.

Proteomics Identification of Differentially Expressed Proteins Associated with Pollen Germination and Tube Growth Reveals Characteristics of Germinated Oryza sativa Pollen

Mature pollen from most plant species is metabolically quiescent; however, after pollination, it germinates quickly and gives rise to a pollen tube to transport sperms into the embryo sac. Because methods for collecting a large amount of in vitro germinated pollen grains for transcriptomics and proteomics studies from model plants of Arabidopsis and rice are not available, molecular information about the germination developmental process is lacking. Here we describe a method for obtaining a large quantity of in vitro germinating rice pollen for proteomics study. Two-dimensional electrophoresis of ∼2300 protein spots revealed 186 that were differentially expressed in mature and germinated pollen. Most showed a changed level of expression, and only 66 appeared to be specific to developmental stages. Furthermore 160 differentially expressed protein spots were identified on mass spectrometry to match 120 diverse protein species. These proteins involve different cellular and metabolic processes with obvious functional skew toward wall metabolism, protein synthesis and degradation, cytoskeleton dynamics, and carbohydrate/energy metabolism. Wall metabolism-related proteins are prominently featured in the differentially expressed proteins and the pollen proteome as compared with rice sporophytic proteomes. Our study also revealed multiple isoforms and differential expression patterns between isoforms of a protein. These results provide novel insights into pollen function specialization.