Narrow leaf 1 (NAL1) regulates leaf shape by affecting cell expansion in rice (Oryza sativa L.).

The narrow leaf1 (nal1) mutant of rice (Oryza sativa L.) exhibits a narrow leaf phenotype. Previous studies have shown that NAL1 modulates leaf size by affecting vein patterning and cell division; however, the underlying mechanism remains unclear. Here, we report that the nal1 mutant shows reduced size of the leaf abaxial epidermal cells and culm parenchyma cells compared with the wild type (WT), indicating that NAL1 also regulates cell expansion. To understand the molecular mechanism of the reduced cell size phenotype, leaves of 40-day-old nal1 mutant and WT seedlings were subjected to RNA-Seq analysis, which has identified 4277 differentially expressed genes (DEGs) between WT and the nal1 mutant. Gene ontology (GO) enrichment analysis revealed a large number of genes down-regulated in the nal1 mutant were involved in cell wall formation. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that NAL1-regulated DEGs, such as ARFs and SAURs, were mapped in auxin signal transduction and auxin-regulated cell expansion pathways. A combination of RNA-Seq analysis and gene expression validation using RT-qPCR suggested that NAL1 is involved in the regulation of auxin-mediated acid growth in rice. These results indicate that, in addition to controlling cell division, NAL1 controls leaf width, at least partially, through its effect on cell expansion, probably via the acid growth mechanism.

VdThit, a Thiamine Transport Protein, Is Required for Pathogenicity of the Vascular Pathogen Verticillium dahliae.

Verticillium dahliae causes a serious wilt disease of important crops and is difficult to control. Few plasma-membrane transport proteins for nutrient acquisition have been identified for this fungus, and their involvement in the disease process is unknown. Here, a plasma-membrane protein, the V. dahliae thiamine transporter protein VdThit, was characterized functionally by deletion of the VdThit gene in V. dahliae. Disruption strains were viable, but growth and conidial germination and production were reduced and virulence was impaired. Interestingly, by supplementing exogenous thiamine, growth, conidiation, and virulence of the VdΔThit mutants were partially restored. Stress-tolerance assays showed that the VdΔThit mutant strains were markedly more susceptible to oxidative stress and UV damage. High-pressure liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS) analyses showed low levels of pyruvate metabolism intermediates acetoin and acetyl coenzyme A (acetyl-CoA) in the VdΔThit mutant strains, suggesting that pyruvate metabolism was suppressed. Expression analysis of VdThit confirmed the importance of VdThit in vegetative growth, reproduction, and invasive hyphal growth. Furthermore, a green fluorescent protein (GFP)-labeled VdΔThit mutant (VdΔThit-7-GFP) was suppressed in initial infection and root colonization, as viewed with light microscopy. Together, these results showed that VdThit plays an indispensable role in the pathogenicity of V. dahliae.

A ku70 null mutant improves gene targeting frequency in the fungal pathogen Verticillium dahliae.

To overcome the challenges met with gene deletion in the plant pathogen Verticillium dahliae, a mutant strain with impaired non-homologous end joining DNA repair was generated to improve targeted gene replacement frequencies. A V. dahliae 991 ΔVdku70 null mutant strain was generated using Agrobacterium tumefaciens-mediated transformation. Despite having impaired non-homologous end joining DNA repair function, the ΔVdku70 strain exhibited normal growth, reproduction capability, and pathogenicity when compared with the wild-type strain. When the ΔVdku70 strain was used to delete 2-oxoglutarate dehydrogenase E2, ferric reductase transmembrane component 3 precursor, and ferric reductase transmembrane component 6 genes, gene replacement frequencies ranged between 22.8 and 34.7% compared with 0.3 and 0.5 % in the wild-type strain. The ΔVdku70 strain will be a valuable tool to generate deletion strains when studying factors that underlie virulence and pathogenesis in this filamentous fungus.

Starch accumulation, activities of key enzyme and gene expression in starch synthesis of wheat endosperm with different starch contents.

In order to investigate starch accumulation, and the enzymes activity changes and the expression levels of genes and their relationships among them at different developmental stages of wheat grain. We choose Annong9912 and E28 were used in the study. During starch accumulating rate and grain filling rate, and there were obvious genotype difference between Annong9912 and E28. Whether low or high starch content of starch content, the accumulation courses of amylopectin, amylose and total starch were well fitted to the logistic equation by relating starch contents against DAP. The simulation parameters revealed that the higher contents of amylopectin and amylose resulted from earlier initiating accumulation time and greater accumulation rate. And amylose, amylopectin and total starch accumulation rate of two wheat cultures were significantly and positively correlated with activities of SBE, SSS and GBSS, but amylose accumulation rate of E28 had no correlation with the activities of SBE. In addition, there were significant correlations among activities of SBE, SSS and GBSS in two wheat cultivars. We speculated that these enzymes proteins may have a coordinating action in starch biosynthesis within the amyloplast, operating as functional multiprotein complexes. And expression levels of enzyme genes demonstrated a single-peak curve, and 12-18 DAP reached their peaks and then began to drop, and all had high expression level in earlier stage of endosperm development, but in E28 were higher than in Annong9912. The GBSS-I transcripts on average were expressed over 60 times more than GBSS-II transcript in E28. SBE, SSS, DBE may control starch synthesis at the transcriptional level, and GBSS-I may control starch synthesis at the post transcriptional level. The expression level of DBE on average was lower than SS-1 and SBE-IIa genes, and similar to SS-III and SBE-IIb genes, but higher than GBSS-I and GBSS-II genes.

Starch accumulation in hulless barley during grain filling.

BACKGROUND: Starch consists of two types of molecules: amylose and amylopectin. The objective of this study was increase understanding about mechanisms related to starch accumulation in hulless barley (Hordeum vulgare L.) grain by measuring temporal changes in (i) grain amylose and amylopectin content, (ii) starch synthase activity, and (iii) the relative expressions of key starch-related genes. RESULTS: The amylopectin/amylose ratio gradually declined in both Beiqing 6 and Kunlun 12. In both cultivars, the activities of adenosine diphosphate glucose pyrophosphorylase, soluble starch synthase (SSS), granule bound starch synthase (GBSS), and starch branching enzyme (SBE) increased steadily during grain filling, reaching their maximums 20-25 days after anthesis. The activities of SSS and SBE were greater in Ganken 5 than in either Beiqing 6 or Kunlun 12. The expression of GBSS I was greater in Beiqing 6 and Kunlun 12 than in Ganken 5. In contrast, the expression of SSS I, SSS II and SBE I was greater in Ganken 5 than in Beiqing 6 and Kunlun 12. The peak in GBSS I expression was later than that of SSS I, SSS II, SBE IIa and SBE IIb. The GBSS I transcript in Kunlun 12 was expressed on average 90 times more than the GBSS II transcript. CONCLUSIONS: The results suggest that SBE and SSS may control starch synthesis at the transcriptional level, whereas GBSS I may control starch synthesis at the post transcriptional level. GBSS I is mainly responsible for amylose synthesis whereas SSS I and SBE II are mainly responsible for amylopectin synthesis in amyloplasts.