Genome-Wide Analysis of the SRPP/REF Gene Family in Taraxacum kok-saghyz Provides Insights into Its Expression Patterns in Response to Ethylene and Methyl Jasmonate Treatments.

Taraxacum kok-saghyz (TKS) is a model plant and a potential rubber-producing crop for the study of natural rubber (NR) biosynthesis. The precise analysis of the NR biosynthesis mechanism is an important theoretical basis for improving rubber yield. The small rubber particle protein (SRPP) and rubber elongation factor (REF) are located in the membrane of rubber particles and play crucial roles in rubber biosynthesis. However, the specific functions of the SRPP/REF gene family in the rubber biosynthesis mechanism have not been fully resolved. In this study, we performed a genome-wide identification of the 10 TkSRPP and 2 TkREF genes' family members of Russian dandelion and a comprehensive investigation on the evolution of the ethylene/methyl jasmonate-induced expression of the SRPP/REF gene family in TKS. Based on phylogenetic analysis, 12 TkSRPP/REFs proteins were divided into five subclades. Our study revealed one functional domain and 10 motifs in these proteins. The SRPP/REF protein sequences all contain typical REF structural domains and belong to the same superfamily. Members of this family are most closely related to the orthologous species T. mongolicum and share the same distribution pattern of SRPP/REF genes in T. mongolicum and L. sativa, both of which belong to the family Asteraceae. Collinearity analysis showed that segmental duplication events played a key role in the expansion of the TkSRPP/REFs gene family. The expression levels of most TkSRPP/REF members were significantly increased in different tissues of T. kok-saghyz after induction with ethylene and methyl jasmonate. These results will provide a theoretical basis for the selection of candidate genes for the molecular breeding of T. kok-saghyz and the precise resolution of the mechanism of natural rubber production.

Genome-Wide Analysis of BBX Gene Family in Three Medicago Species Provides Insights into Expression Patterns under Hormonal and Salt Stresses.

BBX protein is a class of zinc finger transcription factors that have B-box domains at the N-terminus, and some of these proteins contain a CCT domain at the C-terminus. It plays an important role in plant growth, development, and metabolism. However, the expression pattern of BBX genes in alfalfa under hormonal and salt stresses is still unclear. In this study, we identified a total of 125 BBX gene family members by the available Medicago reference genome in diploid alfalfa (Medicago sativa spp. Caerulea), a model plant (M. truncatula), and tetraploid alfalfa (M. sativa), and divided these members into five subfamilies. We found that the conserved motifs of BBXs of the same subfamily reveal similarities. We analyzed the collinearity relationship and duplication mode of these BBX genes and found that the expression pattern of BBX genes is specific in different tissues. Analysis of the available transcriptome data suggests that some members of the BBX gene family are involved in multiple abiotic stress responses, and the highly expressed genes are often clustered together. Furthermore, we identified different expression patterns of some BBX genes under salt, ethylene, salt and ethylene, salicylic acid, and salt and salicylic acid treatments, verified by qRT-PCR, and analyzed the subcellular localization of MsBBX2, MsBBX17, and MsBBX32 using transient expression in tobacco. The results showed that BBX genes were localized in the nucleus. This study systematically analyzed the BBX gene family in Medicago plants, which provides a basis for the study of BBX gene family tolerance to abiotic stresses.

A mutation in Arabidopsis SAL1 alters its in vitro activity against IP3 and delays developmental leaf senescence in association with lower ROS levels.

KEY MESSAGE: Our manuscript is the first to find a link between activity of SAL1/OLD101 against IP3 and plant leaf senescence regulation and ROS levels assigning a potential biological role for IP3. Leaf senescence is a genetically programmed process that limits the longevity of a leaf. We identified and analyzed the recessive Arabidopsis stay-green mutation onset of leaf death 101 (old101). Developmental leaf longevity is extended in old101 plants, which coincided with higher peroxidase activity and decreased H2O2 levels in young 10-day-old, but not 25-day-old plants. The old101 phenotype is caused by a point mutation in SAL1, which encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3' (2'), 5'-bisphosphate nucleotidase activity. SAL1 activity is highly specific for its substrates 3-polyadenosine 5-phosphate (PAP) and inositol 1, 4, 5-trisphosphate (IP3), where it removes the 1-phosphate group from the IP3 second messenger. The in vitro activity of recombinant old101 protein against its substrate IP3 was 2.5-fold lower than that of wild type SAL1 protein. However, the in vitro activity of recombinant old101 mutant protein against PAP remained the same as that of the wild type SAL1 protein. The results open the possibility that the activity of SAL1 against IP3 may affect the redox balance of young seedlings and that this delays the onset of leaf senescence.

Functional analysis of RXLR effectors from the New Zealand kauri dieback pathogen Phytophthora agathidicida.

New Zealand kauri is an ancient, iconic, gymnosperm tree species that is under threat from a lethal dieback disease caused by the oomycete Phytophthora agathidicida. To gain insight into this pathogen, we determined whether proteinaceous effectors of P. agathidicida interact with the immune system of a model angiosperm, Nicotiana, as previously shown for Phytophthora pathogens of angiosperms. From the P. agathidicida genome, we defined and analysed a set of RXLR effectors, a class of proteins that typically have important roles in suppressing or activating the plant immune system. RXLRs were screened for their ability to activate or suppress the Nicotiana plant immune system using Agrobacterium tumefaciens transient transformation assays. Nine P. agathidicida RXLRs triggered cell death or suppressed plant immunity in Nicotiana, of which three were expressed in kauri. For the most highly expressed, P. agathidicida (Pa) RXLR24, candidate cognate immune receptors associated with cell death were identified in Nicotiana benthamiana using RNA silencing-based approaches. Our results show that RXLRs of a pathogen of gymnosperms can interact with the immune system of an angiosperm species. This study provides an important foundation for studying the molecular basis of plant-pathogen interactions in gymnosperm forest trees, including kauri.

Genome draft of the Arabidopsis relative Pachycladon cheesemanii reveals novel strategies to tolerate New Zealand's high ultraviolet B radiation environment.

BACKGROUND: Pachycladon cheesemanii is a close relative of Arabidopsis thaliana and is an allotetraploid perennial herb which is widespread in the South Island of New Zealand. It grows at altitudes of up to 1000 m where it is subject to relatively high levels of ultraviolet (UV)-B radiation. To gain first insights into how Pachycladon copes with UV-B stress, we sequenced its genome and compared the UV-B tolerance of two Pachycladon accessions with those of two A. thaliana accessions from different altitudes. RESULTS: A high-quality draft genome of P. cheesemanii was assembled with a high percentage of conserved single-copy plant orthologs. Synteny analysis with genomes from other species of the Brassicaceae family found a close phylogenetic relationship of P. cheesemanii with Boechera stricta from Brassicaceae lineage I. While UV-B radiation caused a greater growth reduction in the A. thaliana accessions than in the P. cheesemanii accessions, growth was not reduced in one P. cheesemanii accession. The homologues of A. thaliana UV-B radiation response genes were duplicated in P. cheesemanii, and an expression analysis of those genes indicated that the tolerance mechanism in P. cheesemanii appears to differ from that in A. thaliana. CONCLUSION: Although the P. cheesemanii genome shows close similarity with that of A. thaliana, it appears to have evolved novel strategies allowing the plant to tolerate relatively high UV-B radiation.

Drought-induced senescence of Medicago truncatula nodules involves serpin and ferritin to control proteolytic activity and iron levels.

Drought is a major constraint for legume growth and yield. Senescence of nitrogen-fixing nodules is one of the early drought responses and may cause nutrient stress in addition to water stress in legumes. For nodule senescence to function as part of a drought-survival strategy, we propose that the intrinsically destructive senescence process must be tightly regulated. Medicago truncatula protease inhibitor and iron scavenger-encoding genes, possibly involved in controlling nodule senescence, were identified. RNA interference (RNAi) lines were constructed in which expression of a serpin or ferritins was knocked down. Both wild-type and RNAi lines were subjected to drought stress and nodule activity and plant physiological responses were measured. Drought caused M. truncatula to initiate nodule senescence before plant growth was affected and before an increase in papain-like proteolytic activity and free iron levels was apparent. Knock-down expression of serpin6 and ferritins caused increased protease activity, free iron levels, early nodule senescence and reduced plant growth. The results suggest that M. truncatula nodule-expressed serpin6 and ferritins mediate ordered drought-induced senescence by regulating papain-like cysteine protease activity and free iron levels. This strategy may allow the drought-stressed plants to benefit maximally from residual nitrogen fixation and nutrient recovery resulting from break down of macromolecules.

ß-Substituting alanine synthases: roles in cysteine metabolism and abiotic and biotic stress signalling in plants.

Cysteine is required for the synthesis of proteins and metabolites, and is therefore an indispensable compound for growth and development. The ß-substituting alanine synthase (BSAS) gene family encodes enzymes known as O-acetylserine thiol lyases (OASTLs), which carry out cysteine biosynthesis in plants. The functions of the BSAS isoforms have been reported to be crucial in assimilation of S and cysteine biosynthesis, and homeostasis in plants. In this review we explore the functional variation in this classic pyridoxal-phosphate-dependent enzyme family of BSAS isoforms. We discuss how specialisation and divergence in BSAS catalytic activities makes a more dynamic set of biological routers that integrate cysteine metabolism and abiotic and biotic stress signalling in Arabidopsis thaliana (L.) Heynh. and also other species. Our review presents a universal scenario in which enzymes modulating cysteine metabolism promote survival and fitness of the species by counteracting internal and external stress factors.

Transcription of Biotic Stress Associated Genes in White Clover (Trifolium repens L.) Differs in Response to Cyst and Root-Knot Nematode Infection.

The transcription of four members of the Kunitz proteinase inhibitor (KPI) gene family of white clover (Trifolium repens L.), designated as Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5, was investigated at both local infection (roots) and systemic (leaf tissue) sites in white clover in response to infection with the clover root knot nematode (CRKN) Meloidogyne trifoliophila and the clover cyst nematode (CCN) Heterodera trifolii. Invasion by the CRKN resulted in a significant decrease in transcript abundance of Tr-KPI4 locally at both 4 days post-infection (dpi) and at 8 dpi, and an increase in transcription of Tr-KPI1 systemically at 8 dpi. In contrast, an increase in transcript abundance of all four Tr-KPI genes locally at 4 and 8 dpi, and an increase of Tr-KPI1, Tr-KPI2, and Tr-KPI5 at 8 dpi systemically was observed in response to infection with the CCN. Challenge of a resistant (R) genotype and a susceptible (S) genotype of white clover with the CCN revealed a significant increase in transcript abundance of all four Tr-KPI genes locally in the R genotype, while an increase in abundance of only Tr-KPI1, Tr-KPI2, and Tr-KPI5 was observed in the S genotype, and only at 4 dpi. The transcript abundance of a member of the1-AMINOCYCLOPROPANE-1-CARBOXYLATE (ACC) SYNTHASE gene family from white clover (Tr-ACS1) was significantly down-regulated locally in response to CRKN infection at 4 and 8 dpi and at 4 dpi, systemically, while abundance increased locally and systemically at 8 dpi in response to CCN challenge. Conversely, the abundance of the jasmonic acid (JA) signalling gene, CORONATINE-INSENSITIVE PROTEIN 1 from white clover (Tr-COI1) increased significantly at 8 dpi locally in response to CRKN infection, but decreased at 8 dpi in response to CCN infection. The significance of this differential regulation of transcription is discussed with respect to differences in infection strategy of the two nematode species.

Knock-down of transcript abundance of a family of Kunitz proteinase inhibitor genes in white clover (Trifolium repens) reveals a redundancy and diversity of gene function.

The transcriptional regulation of four phylogenetically distinct members of a family of Kunitz proteinase inhibitor (KPI) genes isolated from white clover (Trifolium repens; designated Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5) has been investigated to determine their wider functional role. The four genes displayed differential transcription during seed germination, and in different tissues of the mature plant, and transcription was also ontogenetically regulated. Heterologous over-expression of Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5 in Nicotiana tabacum retarded larval growth of the herbivore Spodoptera litura, and an increase in the transcription of the pathogenesis-related genes PR1 and PR4 was observed in the Tr-KPI1 and Tr-KPI4 over-expressing lines. RNA interference (RNAi) knock-down lines in white clover displayed significantly altered vegetative growth phenotypes with inhibition of shoot growth and a stimulation of root growth, while knock-down of Tr-KPI1, Tr-KPI2 and Tr-KPI5 transcript abundance also retarded larval growth of S. litura. Examination of these RNAi lines revealed constitutive stress-associated phenotypes as well as altered transcription of cellular signalling genes. These results reveal a functional redundancy across members of the KPI gene family. Further, the regulation of transcription of at least one member of the family, Tr-KPI2, may occupy a central role in the maintenance of a cellular homeostasis.

Activation of R-mediated innate immunity and disease susceptibility is affected by mutations in a cytosolic O-acetylserine (thiol) lyase in Arabidopsis.

O-acetylserine (thiol) lyases (OASTLs) are evolutionarily conserved proteins among many prokaryotes and eukaryotes that perform sulfur acquisition and synthesis of cysteine. A mutation in the cytosolic OASTL-A1 protein ONSET OF LEAF DEATH3 (OLD3) was previously shown to reduce the OASTL activity of the old3-1 protein in vitro and cause auto-necrosis in specific Arabidopsis accessions. Here we investigated why a mutation in this protein causes auto-necrosis in some but not other accessions. The auto-necrosis was found to depend on Recognition of Peronospora Parasitica 1 (RPP1)-like disease resistance R gene(s) from an evolutionarily divergent R gene cluster that is present in Ler-0 but not the reference accession Col-0. RPP1-like gene(s) show a negative epistatic interaction with the old3-1 mutation that is not linked to reduced cysteine biosynthesis. Metabolic profiling and transcriptional analysis further indicate that an effector triggered-like immune response and metabolic disorder are associated with auto-necrosis in old3-1 mutants, probably activated by an RPP1-like gene. However, the old3-1 protein in itself results in largely neutral changes in primary plant metabolism, stress defence and immune responses. Finally, we showed that lack of a functional OASTL-A1 results in enhanced disease susceptibility against infection with virulent and non-virulent Pseudomonas syringae pv. tomato DC3000 strains. These results reveal an interaction between the cytosolic OASTL and components of plant immunity.

A mutation in the cytosolic O-acetylserine (thiol) lyase induces a genome-dependent early leaf death phenotype in Arabidopsis.

BACKGROUND: Cysteine is a component in organic compounds including glutathione that have been implicated in the adaptation of plants to stresses. O-acetylserine (thiol) lyase (OAS-TL) catalyses the final step of cysteine biosynthesis. OAS-TL enzyme isoforms are localised in the cytoplasm, the plastids and mitochondria but the contribution of individual OAS-TL isoforms to plant sulphur metabolism has not yet been fully clarified. RESULTS: The seedling lethal phenotype of the Arabidopsis onset of leaf death3-1 (old3-1) mutant is due to a point mutation in the OAS-A1 gene, encoding the cytosolic OAS-TL. The mutation causes a single amino acid substitution from Gly162 to Glu162, abolishing old3-1 OAS-TL activity in vitro. The old3-1 mutation segregates as a monogenic semi-dominant trait when backcrossed to its wild type accession Landsberg erecta (Ler-0) and the Di-2 accession. Consistent with its semi-dominant behaviour, wild type Ler-0 plants transformed with the mutated old3-1 gene, displayed the early leaf death phenotype. However, the old3-1 mutation segregates in an 11:4:1 (wild type: semi-dominant: mutant) ratio when backcrossed to the Colombia-0 and Wassilewskija accessions. Thus, the early leaf death phenotype depends on two semi-dominant loci. The second locus that determines the old3-1 early leaf death phenotype is referred to as odd-ler (for old3 determinant in the Ler accession) and is located on chromosome 3. The early leaf death phenotype is temperature dependent and is associated with increased expression of defence-response and oxidative-stress marker genes. Independent of the presence of the odd-ler gene, OAS-A1 is involved in maintaining sulphur and thiol levels and is required for resistance against cadmium stress. CONCLUSIONS: The cytosolic OAS-TL is involved in maintaining organic sulphur levels. The old3-1 mutation causes genome-dependent and independent phenotypes and uncovers a novel function for the mutated OAS-TL in cell death regulation.

Ethylene-induced leaf senescence depends on age-related changes and OLD genes in Arabidopsis.

Ethylene can only induce senescence in leaves that have reached a defined age. Thus, ethylene-induced senescence depends on age-related changes (ARCs) of individual leaves. The relationship between ethylene and age in the induction of leaf senescence was tested in Arabidopsis Ler-0, Col-0, and Ws-0 accessions as well as in eight old (onset of leaf death) mutants, isolated from the Ler-0 background. Plants with a constant final age of 24 d were exposed to ethylene for 3-16 d. The wild-type accessions showed a common response to the ethylene treatment. Increasing ethylene treatments of 3-12 d caused an increase in the number of yellow leaves. However, an ethylene exposure time of 16 d resulted in a decrease in the amount of yellowing. Thus, ethylene can both positively and negatively influence ARCs and the subsequent induction of leaf senescence, depending on the length of the treatment. The old mutants showed altered responses to the ethylene treatments. old1 and old11 were hypersensitive to ethylene in the triple response assay and a 12-d ethylene exposure resulted in a decrease in the amount of yellow leaves. The other six mutants did not show a decrease in yellow leaves with an ethylene treatment of 16 d. The results revealed that the effect of ethylene on the induction of senescence can be modified by at least eight genes.