Transcription factor gene TuGTγ-3 is involved in the stripe rust resistance in Triticum urartu.

Wheat stripe rust, caused by Puccinia striiformis West. f. sp. tritici Eriks. ＆Henn. (Pst), is a serious fungal disease. Identification of new genes associate with stripe rust resistance is important for developing disease resistant wheat cultivars and studying the mechanism of disease resistance. Trihelix is a plant specific transcription factor family, which is involved in regulation of growth and development, morphogenesis, and response to stresses. So far, no study reports on the relationship between the Trihelix family and wheat stripe rust. In this study, a gene in the GTγ subfamily of Trihelix family, designated TuGTγ-3, was cloned from Triticum urartu Tum. (2n=2x=14, AA). The results of sequencing demonstrated that TuGTγ-3 gene consisted of a complete open reading frame (ORF), and its coding sequence was 1329 bp in length, which encoded a protein with 442 amino acids. The predicted molecular weight of this protein was 50.31 kDa and the theoretical isoelectric point was 6.12. Bioinformatic analysis revealed that TuGTγ-3 protein had a monopartite nuclear localization signal (GLPMQKKMRYT), and had neither transmembrane domain nor signal peptide. The conserved trihelix domain, the fourth α-helix and the CC domain were located in the regions of Q115?R187, F234?Y241 and K362?K436, respectively. Dissection of secondary structure showed that TuGTγ-3 protein comprised of 43.89% α-helix, 9.51% extended strand, 9.95% ß-turn and 36.65% random coil structures. Based on the BLAST search against the genome database of common wheat from IWGSC, TuGTγ-3 was located on the long arm of chromosome 5A. Transient expression experiment using onion inner epidermal cell showed that the fusion protein TuGTγ-3-GFP distributed mainly in nuclear and slightly in cytoplasm. Expression profiles in different organs indicated that expression level of TuGTγ-3 was much higher in leaves than that in roots or leaf sheaths, and the expression in leaves was extremely up-regulated by infection of the Pst race CYR32. Furthermore, the BSMV-VIGS experiment demonstrated that the transcription factor TuGTγ-3 positively regulated resistance to stripe rust in T. urartu.

Genome-wide analysis of the MADS-box gene family in Brachypodium distachyon.

MADS-box genes are important transcription factors for plant development, especially floral organogenesis. Brachypodium distachyon is a model for biofuel plants and temperate grasses such as wheat and barley, but a comprehensive analysis of MADS-box family proteins in Brachypodium is still missing. We report here a genome-wide analysis of the MADS-box gene family in Brachypodium distachyon. We identified 57 MADS-box genes and classified them into 32 MIKC(c)-type, 7 MIKC*-type, 9 Mα, 7 Mß and 2 Mγ MADS-box genes according to their phylogenetic relationships to the Arabidopsis and rice MADS-box genes. Detailed gene structure and motif distribution were then studied. Investigation of their chromosomal localizations revealed that Brachypodium MADS-box genes distributed evenly across five chromosomes. In addition, five pairs of type II MADS-box genes were found on synteny blocks derived from whole genome duplication blocks. We then performed a systematic expression analysis of Brachypodium MADS-box genes in various tissues, particular floral organs. Further detection under salt, drought, and low-temperature conditions showed that some MADS-box genes may also be involved in abiotic stress responses, including type I genes. Comparative studies of MADS-box genes among Brachypodium, rice and Arabidopsis showed that Brachypodium had fewer gene duplication events. Taken together, this work provides useful data for further functional studies of MADS-box genes in Brachypodium distachyon.

Arabidopsis sucrose transporter SUT4 interacts with cytochrome b5-2 to regulate seed germination in response to sucrose and glucose.

It remains unknown whether a sucrose transporter mediates sugar signaling. Here, we report that the Arabidopsis (Arabidopsis thaliana) sucrose transporter SUT4 interacts with five members of the Arabidopsis cytochrome b5 (Cyb5) family, and sucrose represses the interaction between SUT4 and a Cyb5 member Cyb5-2/A. We observed that down-regulation of SUT4 and three cytochrome b5 members (Cyb5-2, Cyb5-4, and Cyb5-6) confers the sucrose- and glucose-insensitive phenotypes in the sucrose/glucose-induced inhibition of seed germination. The sut4 cyb5-2 double mutant displays slightly stronger sucrose/glucose-insensitive phenotypes than either the sut4 or cyb5-2 single mutant. We showed that the SUT4/Cyb5-2-mediated signaling in the sucrose/glucose-induced inhibition of seed germination does not require ABA or the currently known ABI2/ABI4/ABI5-mediated signaling pathway(s). These data provide evidence that the sucrose transporter SUT4 interacts with Cyb5 to positively mediate sucrose and glucose signaling in the sucrose/glucose-induced inhibition of seed germination.

Apple sucrose transporter SUT1 and sorbitol transporter SOT6 interact with cytochrome b5 to regulate their affinity for substrate sugars.

Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 --> proline in MdSUT1 and leucine-117 --> proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability.

Ubiquitous distribution and different subcellular localization of sorbitol dehydrogenase in fruit and leaf of apple.

NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), a key enzyme in sorbitol metabolism, plays an important role in regulating sink strength and determining the quality of apple fruit. Understanding the tissue and subcellular localization of NAD-SDH is helpful for understanding sorbitol metabolism in the apple. In this study, two NAD-SDH cDNA sequences were isolated from apple fruits (Malus domestica Borkh cv. Starkrimson) and named MdSDH5 and MdSDH6. Immunohistochemical analysis revealed that NAD-SDH is distributed in both the flesh and the vascular tissue of the fruit, and the vascular tissue and mesophyll tissue in the young and old leaves, indicating that it is a ubiquitous protein expressed in both sink and source organs. Immunogold electron microscopy analysis demonstrated that NAD-SDH is localized mainly in the cytoplasm and chloroplast of the fruit and leaves. The chloroplast localization of NAD-SDH was confirmed by the transient expression of MdSDH5-GFP and MdSDH6-GFP in the mesophyll protoplast of Arabidopsis. NAD-SDH was also found in electron opaque deposits of vacuoles in young and mature leaves. These data show that NAD-SDH has different subcellular localizations in fruit and leaves, indicating that it might play a different role in sorbitol metabolism in different tissues of apple.

Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis.

Many biochemical approaches show functions of calcium-dependent protein kinases (CDPKs) in abscisic acid (ABA) signal transduction, but molecular genetic evidence linking defined CDPK genes with ABA-regulated biological functions at the whole-plant level has been lacking. Here, we report that ABA stimulated two homologous CDPKs in Arabidopsis thaliana, CPK4 and CPK11. Loss-of-function mutations of CPK4 and CPK11 resulted in pleiotropic ABA-insensitive phenotypes in seed germination, seedling growth, and stomatal movement and led to salt insensitivity in seed germination and decreased tolerance of seedlings to salt stress. Double mutants of the two CDPK genes had stronger ABA- and salt-responsive phenotypes than the single mutants. CPK4- or CPK11-overexpressing plants generally showed inverse ABA-related phenotypes relative to those of the loss-of-function mutants. Expression levels of many ABA-responsive genes were altered in the loss-of-function mutants and overexpression lines. The CPK4 and CPK11 kinases both phosphorylated two ABA-responsive transcription factors, ABF1 and ABF4, in vitro, suggesting that the two kinases may regulate ABA signaling through these transcription factors. These data provide in planta genetic evidence for the involvement of CDPK/calcium in ABA signaling at the whole-plant level and show that CPK4 and CPK11 are two important positive regulators in CDPK/calcium-mediated ABA signaling pathways.

The Mg-chelatase H subunit is an abscisic acid receptor.

Abscisic acid (ABA) is a vital phytohormone that regulates mainly stomatal aperture and seed development, but ABA receptors involved in these processes have yet to be determined. We previously identified from broad bean an ABA-binding protein (ABAR) potentially involved in stomatal signalling, the gene for which encodes the H subunit of Mg-chelatase (CHLH), which is a key component in both chlorophyll biosynthesis and plastid-to-nucleus signalling. Here we show that Arabidopsis ABAR/CHLH specifically binds ABA, and mediates ABA signalling as a positive regulator in seed germination, post-germination growth and stomatal movement, showing that ABAR/CHLH is an ABA receptor. We show also that ABAR/CHLH is a ubiquitous protein expressed in both green and non-green tissues, indicating that it might be able to perceive the ABA signal at the whole-plant level.

A shift of Phloem unloading from symplasmic to apoplasmic pathway is involved in developmental onset of ripening in grape berry.

It remains unclear whether the phloem unloading pathway alters to adapt to developmental transition in fleshy fruits that accumulate high level of soluble sugars. Using a combination of electron microscopy, transport of the phloem-mobile symplasmic tracer carboxyfluorescein, movement of the companion cell-expressed and the green fluorescent protein-tagged viral movement protein, and assays of the sucrose cleavage enzymes, the pathway of phloem unloading was studied in the berries of a hybrid grape (Vitis vinifera x Vitis labrusca). Structural investigations showed that the sieve element-companion cell complex is apparently symplasmically connected through plasmodesmata with surrounding parenchyma cells throughout fruit development, though a small portion of plasmodesmata are apparently blocked in the ripening stage. Both carboxyfluorescein and the green fluorescent protein-tagged viral movement protein were released from the functional phloem strands during the early and middle stages of fruit development, whereas the two symplasmic tracers were confined to the phloem strands during the late stage. This reveals a shift of phloem unloading from symplasmic to apoplasmic pathway during fruit development. The turning point of the phloem unloading pathways was further shown to be at or just before onset of ripening, an important developmental checkpoint of grape berry. In addition, the levels of both the expression and activities of cell wall acid invertase increased around the onset of ripening and reached a high level in the late stage, providing further evidence for an operation of the apoplasmic unloading pathway after onset of ripening. These data demonstrate clearly the occurrence of an adaptive shift of phloem unloading pathway to developmental transition from growing phase to ripening in grape berry.