Young-Woo Nam

A novel Δ1-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation

Proline accumulates in environmentally stressed plant cells including those of legume roots and nodules, but how its level is regulated is poorly understood. Δ(1)-Pyrroline-5-carboxylate synthetase (P5CS), the committed-step enzyme of proline biosynthesis, is encoded by two duplicated genes in many plants. Here, we isolated MtP5CS3, a third gene, from Medicago truncatula, whose predicted polypeptide sequence is highly similar to those of previously isolated MtP5CS1 and MtP5CS2 except an extra amino-terminal segment. MtP5CS3 was strongly expressed under salinity and drought in shoots and nodulating roots, while MtP5CS1 was constitutive and MtP5CS2 induced by abscisic acid. Under salinity, MtP5CS3 promoter was more active than those of MtP5CS1 and MtP5CS2, as shown by GUS fusions. Translationally fused MtP5CS1-GFP was localized in the cytoplasm, whereas significant proportions of MtP5CS2-GFP and MtP5CS3-GFP were co-localized with rubisco small subunit protein-fused RFP in transformed hairy root cells. Under salinity, RNA silencing of MtP5CS1 or MtP5CS2 strongly induced MtP5CS3 expression, while that of MtP5CS3 decreased free proline content and nodule number. Consistently, Mtp5cs3, a loss-of-function mutant, accumulated much less proline, formed fewer nodules, and fixed nitrogen significantly less efficiently than the wild type under salinity. Thus, MtP5CS3 plays a critical role in regulating stress-induced proline accumulation during symbiotic nitrogen fixation.

Molecular cytogenetic mapping of Cucumis sativus and C. melo using highly repetitive DNA sequences

Chromosomes often serve as one of the most important molecular aspects of studying the evolution of species. Indeed, most of the crucial mutations that led to differentiation of species during the evolution have occurred at the chromosomal level. Furthermore, the analysis of pachytene chromosomes appears to be an invaluable tool for the study of evolution due to its effectiveness in chromosome identification and precise physical gene mapping. By applying fluorescence in situ hybridization of 45S rDNA and CsCent1 probes to cucumber pachytene chromosomes, here, we demonstrate that cucumber chromosomes 1 and 2 may have evolved from fusions of ancestral karyotype with chromosome number n = 12. This conclusion is further supported by the centromeric sequence similarity between cucumber and melon, which suggests that these sequences evolved from a common ancestor. It may be after or during speciation that these sequences were specifically amplified, after which they diverged and specific sequence variants were homogenized. Additionally, a structural change on the centromeric region of cucumber chromosome 4 was revealed by fiber-FISH using the mitochondrial-related repetitive sequences, BAC-E38 and CsCent1. These showed the former sequences being integrated into the latter in multiple regions. The data presented here are useful resources for comparative genomics and cytogenetics of Cucumis and, in particular, the ongoing genome sequencing project of cucumber.

Microsatellite-based analysis of genetic diversity in 91 commercial Brassica oleracea L. cultivars belonging to six varietal groups

Brassica oleracea L. includes various types of important vegetables that show extremely diverse phenotypes. To elucidate the genetic diversity and relationships among commercial cultivars derived by different companies throughout the world, we characterized the diversity and genetic structure of 91 commercial B. oleracea cultivars belonging to six varietal groups, including cabbage, broccoli, cauliflower, kohlrabi, kale and kai-lan. We used 69 polymorphic microsatellite markers showing a total of 359 alleles with an average number of 5.20 alleles per locus. Polymorphism information content (PIC) values ranged from 0.06 to 0.73, with an average of 0.40. Among the six varietal groups, kohlrabi cultivars exhibited the highest heterozygosity level, whereas kale cultivars showed the lowest. Based on genetic similarity values, an UPGMA clustering dendrogram and a two-dimensional scale diagram (PCoA) were generated to analyze genetic diversity. The cultivars were clearly separated into six different clusters with a tendency to cluster into varietal groups. Model-based structure analysis revealed six genetic groups, in which cabbage cultivars were divided into two subgroups that were differentiated by their head shape, whereas cauliflower and kai-lan cultivars clustered together into a single group. Furthermore, we identified 18 SSR markers showing 27 unique alleles specific to only one cultivar that can be used to discriminate 22 cultivars from the others. Our phylogenetic and population structure analysis presents new insights into the genetic structure and relationships among 91 B. oleracea cultivars and provides valuable information for breeding of B. oleracea species. In addition, we demonstrate the utility of SSR markers as a powerful tool for discriminating between the cultivars. The SSR markers described herein will also be helpful for Distinctness, Uniformity and Stability (DUS) test of new cultivars.

Isolation and characterization of cDNA clones expressed under male sex expression conditions in a monoecious cucumber plant (Cucumis sativus L. cv. Winter Long)

In order to clone sex determination genes, both differential screening of a cDNA library and differential display reverse transcription PCR (DDRT-PCR) were conducted, using apex tissues from a Korean cucumber cultivar that exhibited different sex expression responses to differing photoperiods. Two genes (CsM1 and CsM2) expressed only under specific male expression conditions were then cloned via differential screening of a cDNA library and designated CsCYR (Cucumis sativus cyclin-related) and CsCYP (C. sativus cyclophilin). Both genes appear to be single-copy genes, and both express under male-expressing SD (short-day) conditions. CsM10 (C. sativus male specific clone 10) was isolated via DDRT-PCR, and was expressed preferentially under male expression conditions. The CsM10 gene encodes a non-coding RNA, and harbors a 179bp-conserved sequence, found in three genes which have been classified as a biotic stress signal-associated non-translatable RNA (or non-coding RNA) group. The CsM10 gene is also a single-copy gene, which is localized in the telomeric region of chromosome 6, and its expression is different in different tissues, developmental stages, and photoperiods. Based on the obtained sequence structures and expression patterns of CsM10 and its homologues, we suggest that CsM10 function appears to occur at the RNA level.

Genomic organization and differential expression of two polygalacturonase-inhibiting protein genes fromMedicago truncatula

Two adjacent polygalacturonase-inhibiting protein (PGIP) genes were characterized from the model legumeMedicago truncatula. MtPGIP1 andMtPGIP2 were isolated from a single bacterial artificial chromosome clone identified from library-screening with cDNA probes. Ten and nine characteristic stretches of leucine-rich repeats, respectively, were identified from the predicted MtPCIP1 and MtPGIP2, showing 58% sequence identity at the amino acid level. TheseMtPGIP genes are likely present as a small gene family. Transcripts encoding MtPGIP1 were expressed highly in the flowers and at low levels in the roots and stems, whereas those encoding MtPGIP2 were not detected in any untreated organs. Inoculation of theM. truncatula cultivar “Jemalong” with the pathogenic fungusColletotrichum trifolii induced a hypersensitive response and the expression of both genes. The two genes were also expressed in response to the application of jasmonic acid, although mechanical wounding induced onlyMtPGIP1 and salicylic acid induced neither. Abiotic stresses, such as high-salt, cold, or drought, induced the expression ofMtPGIP1, whereas low-temperature stress inducedMtPGIP1 only. Consistent with these observations, sequence elements specific to plant defense and stress responses were identified, in varying numbers, from the putative promoter regions of the two genes. Furthermore, supportive of their putative functional roles, bacterially expressed recombinant MtPGIP1 and MtPGIP2 inhibited fungal polygalacturonase activity. Therefore, these results suggest thatMtPGIP1 andMtPGIP2, copies that presumably arose from duplication, are regulated by separate signaling pathways and likely play roles in response to pathogenic and environmental stresses.

Single or Multiple Gene Silencing Directed by U6 Promoter-Based shRNA Vectors Facilitates Efficient Functional Genome Analysis in Medicago truncatula

Short hairpin RNA (shRNA) expression vectors are an effective means to deliver double-stranded RNA that mediates sequence-specific cleavage of target transcripts in RNA interference (RNAi). Previously, we constructed shRNA expression vectors based on U6 small nuclear RNA (snRNA) gene promoters of Medicago truncatula. To facilitate screening of shRNA candidates, a protoplast-based dual luciferase assay system was developed, which consists of a shRNA-expressing effector plasmid and a target gene-carrying reporter plasmid. RNAi efficiency of a given shRNA could be measured by comparing two luciferase activities derived from transcripts with an intact or interrupted 3′ untranslated region as a result of RNAi. Using a multiple U6-shRNA cassette plasmid that enables targeting of multiple genes simultaneously, the expression of two Δ1-pyrroline-5-carboxylate synthetase genes (MtP5CS1 and MtP5CS2) was silenced with a U6 plasmid carrying two 27-nucleotide-stem shRNA cassettes targeting each gene. The decreased transcript levels were accompanied by decreased protein levels and decreased free proline contents. To examine possible U6-shRNA-mediated RNAi in nodules, two remorin-encoding MtREM genes were targeted by single and double U6-shRNA plasmids. Silencing of MtREM2.2, an essential nodulation gene, MtREM1.1, an uncharacterized gene, or both inhibited nodule formation in transgenic roots. Moreover, M. truncatula roots transformed with a suppressor-overexpresser construct wherein MtREM2.2-targeting U6-shRNA and GmREM2.3-overexpressing cassettes were placed tandemly showed decreased MtREM2.2 transcripts yet survived nitrogen-free medium, indicating complementation of MtREM2.2 with the overexpressed soybean gene. These results demonstrate that U6 promoter-based shRNA-expressing plasmids are valuable for multiple gene functional analyses in protoplasts and nodules of M. truncatula.

Physiological and metabolomic analysis of a knockout mutant suggests a critical role of MtP5CS3 gene in osmotic stress tolerance of Medicago truncatula

In the model legume Medicago truncatula, Δ1-pyrroline-5-carboxylate synthetase (P5CS), the rate-limiting enzyme of proline biosynthesis, is encoded by three closely related genes, MtP5CS1, MtP5CS2, and MtP5CS3. While MtP5CS1 is constitutively expressed, MtP5CS2 and MtP5CS3 are induced by adverse environmental conditions, of which MtP5CS3 is prevalently expressed during drought and salinity stresses. Mtp5cs3, a transposon (Tnt1) insertion mutant of MtP5CS3 that cannot synthesize a mature protein, showed decreased proline accumulation and increased sensitivity to salinity, drought, and low water potential stresses, as evidenced by decreased seedling growth and chlorophyll content and increased hydrogen peroxide content. These defective phenotypes were complemented by externally supplied proline or ectopically expressed cDNA to the wild-type gene (MtP5CS3). Gas chromatography–mass spectrometry-based analysis of soluble metabolites revealed that some major metabolites contributing to osmotolerance, including certain amino acids, sugars, and polyols, accumulated more abundantly in the Mtp5cs3 roots than in the wild type, whereas a few other amino acids accumulated less during drought and salinity stresses. While such metabolic reconfiguration apparently fell short of compensating for proline deficiency in Mtp5cs3, overexpression of MtP5CS3 significantly increased tolerance of M. truncatula to salinity and low water potential stress. Thus, MtP5CS3 plays a crucial role in proline accumulation and osmotic stress tolerance of M. truncatula. Manipulation of this predominant proline biosynthetic gene will facilitate the development of environmentally stable legume crops.