Ruijun Duan

Interactive Effects of Cytokinins, Light, and Sucrose on the Phenotypes and the Syntheses of Anthocyanins and Lignins in Cytokinin Overproducing Transgenic Arabidopsis

Elevated cytokinins levels in vivo resulted in both enlarged and retarded growth phenotypes. Cytokinin levels were similar in both phenotypic groups. The ratio of the two phenotypes could be altered by changing the sucrose concentration in MS medium or the light intensity. Increased cytokinins in ipt:: GUS activated Arabidopsis induced the syntheses of anthocyanins and lignins, while light speeded up the anthocyanin accumulation. The endogenous increase in cytokinins and exogenous addition of sucrose in MS medium caused similar effects on the formation of anthocyanins and lignins in shoot, especially in the shoot apical meristem. Cytokinins induced CHS transcription in ipt:::GUS-activated Arabidopsis seedlings in the vegetative growth period; the transcription of CHS was low in ipt inactive plants and in wild-type Columbia. Cytokinins were capable of influencing either the synthesis or distribution of chlorophylls, but they did not alter cab and rbcS transcriptions in light-grown seedlings.

Isopentenyl transferase gene (ipt) downstream transcriptionally fused with gene expression improves the growth of transgenic plants

This research reports a promising approach to increase a plant’s physiological cytokinin content. This approach also enables the increase to play a role in plant growth and development by introducing the ipt gene to downstream transcriptionally fuse with other genes under the control of a CaMV35S promoter, in which the ipt gene is far from the 35S promoter. According to Kozak’s ribosome screening model, expression of the ipt gene is reduced by the terminal codon of the first gene and the internal untranslated nucleotides between the fused genes. In the transgenic plants pVKH35S-GUS-ipt, pVKH35S-AOC-ipt, and pVKH35S-AtGolS2-ipt, cytokinins were increased only two to threefold, and the plants grew more vigorously than the pVKH35S-AOC or pVKH35S-AtGolS2 transgenic plants lacking the ipt gene. The vigorous growth was reflected in rapid plant growth, a longer flowering period, a greater number of flowers, more seed product, and increased chlorophyll synthesis. The AOC and AtGolS2 genes play a role in a plant’s tolerance of salt or cold, respectively. When the ipt gene transcriptionally fuses with AOC or AtGolS2 in the frame of AOC-ipt and AtGolS2-ipt, slight cytokinin increases were obtained in their transgenic plants; furthermore, those increases played a positive role in improvements of plant growth. Notably, an increased cytokinin volume at the physiological level, in concert with AtGolS2 expression, enhances a plant’s tolerance to cold.

SpAHA1 and SpSOS1 coordinate in transgenic yeast to improve salt tolerance

In plant cells, the plasma membrane Na+/H+ antiporter SOS1 (salt overly sensitive 1) mediates Na+ extrusion using the proton gradient generated by plasma membrane H+-ATPases, and these two proteins are key plant halotolerance factors. In the present study, two genes from Sesuvium portulacastrum, encoding plasma membrane Na+/H+ antiporter (SpSOS1) and H+-ATPase (SpAHA1), were cloned. Localization of each protein was studied in tobacco cells, and their functions were analyzed in yeast cells. Both SpSOS1 and SpAHA1 are plasma membrane-bound proteins. Real-time polymerase chain reaction (PCR) analyses showed that SpSOS1 and SpAHA1 were induced by salinity, and their expression patterns in roots under salinity were similar. Compared with untransformed yeast cells, SpSOS1 increased the salt tolerance of transgenic yeast by decreasing the Na+ content. The Na+/H+ exchange activity at plasma membrane vesicles was higher in SpSOS1-transgenic yeast than in the untransformed strain. No change was observed in the salt tolerance of yeast cells expressing SpAHA1 alone; however, in yeast transformed with both SpSOS1 and SpAHA1, SpAHA1 generated an increased proton gradient that stimulated the Na+/H+ exchange activity of SpSOS1. In this scenario, more Na+ ions were transported out of cells, and the yeast cells co-expressing SpSOS1 and SpAHA1 grew better than the cells transformed with only SpSOS1 or SpAHA1. These findings demonstrate that the plasma membrane Na+/H+ antiporter SpSOS1 and H+-ATPase SpAHA1 can function in coordination. These results provide a reference for developing more salt-tolerant crops via co-transformation with the plasma membrane Na+/H+ antiporter and H+-ATPase.

Structure, Expression, and Functional Analysis of the Hexokinase Gene Family in Cassava

Hexokinase (HXK) proteins play important roles in catalyzing hexose phosphorylation and sugar sensing and signaling. To investigate the roles of HXKs in cassava tuber root development, seven HXK genes (MeHXK1–7) were isolated and analyzed. A phylogenetic analysis revealed that the MeHXK family can be divided into five subfamilies of plant HXKs. MeHXKs were clearly divided into type A (MeHXK1) and type B (MeHXK2–7) based on their N-terminal sequences. MeHXK1–5 all had typical conserved regions and similar protein structures to the HXKs of other plants; while MeHXK6–7 lacked some of the conserved regions. An expression analysis of the MeHXK genes in cassava organs or tissues demonstrated that MeHXK2 is the dominant HXK in all the examined tissues (leaves, stems, fruits, tuber phloems, and tuber xylems). Notably, the expression of MeHXK2 and the enzymatic activity of HXK were higher at the initial and expanding tuber stages, and lower at the mature tuber stage. Furthermore, the HXK activity of MeHXK2 was identified by functional complementation of the HXK-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). The gene expression and enzymatic activity of MeHXK2 suggest that it might be the main enzyme for hexose phosphorylation during cassava tuber root development, which is involved in sucrose metabolism to regulate the accumulation of starch.

Identification, Expression, and Functional Analysis of the Fructokinase Gene Family in Cassava

Fructokinase (FRK) proteins play important roles in catalyzing fructose phosphorylation and participate in the carbohydrate metabolism of storage organs in plants. To investigate the roles of FRKs in cassava tuber root development, seven FRK genes (MeFRK1–7) were identified, and MeFRK1–6 were isolated. Phylogenetic analysis revealed that the MeFRK family genes can be divided into α (MeFRK 1, 2, 6, 7) and β (MeFRK 3, 4, 5) groups. All the MeFRK proteins have typical conserved regions and substrate binding residues similar to those of the FRKs. The overall predicted three-dimensional structures of MeFRK1–6 were similar, folding into a catalytic domain and a β-sheet ‘‘lid” region, forming a substrate binding cleft, which contains many residues involved in the binding to fructose. The gene and the predicted three-dimensional structures of MeFRK3 and MeFRK4 were the most similar. MeFRK1–6 displayed different expression patterns across different tissues, including leaves, stems, tuber roots, flowers, and fruits. In tuber roots, the expressions of MeFRK3 and MeFRK4 were much higher compared to those of the other genes. Notably, the expression of MeFRK3 and MeFRK4 as well as the enzymatic activity of FRK were higher at the initial and early expanding tuber stages and were lower at the later expanding and mature tuber stages. The FRK activity of MeFRK3 and MeFRK4 was identified by the functional complementation of triple mutant yeast cells that were unable to phosphorylate either glucose or fructose. The gene expression and enzymatic activity of MeFRK3 and MeFRK4 suggest that they might be the main enzymes in fructose phosphorylation for regulating the formation of tuber roots and starch accumulation at the tuber root initial and expanding stages.

Isolation and Characterization of Ftsz Genes in Cassava

The filamenting temperature-sensitive Z proteins (FtsZs) play an important role in plastid division. In this study, three FtsZ genes were isolated from the cassava genome, and named MeFtsZ1, MeFtsZ2-1, and MeFtsZ2-2, respectively. Based on phylogeny, the MeFtsZs were classified into two groups (FtsZ1 and FtsZ2). MeFtsZ1 with a putative signal peptide at N-terminal, has six exons, and is classed to FtsZ1 clade. MeFtsZ2-1 and MeFtsZ2-2 without a putative signal peptide, have seven exons, and are classed to FtsZ2 clade. Subcellular localization found that all the three MeFtsZs could locate in chloroplasts and form a ring in chloroplastids. Structure analysis found that all MeFtsZ proteins contain a conserved guanosine triphosphatase (GTPase) domain in favor of generate contractile force for cassava plastid division. The expression profiles of MeFtsZ genes by quantitative reverse transcription-PCR (qRT-PCR) analysis in photosynthetic and non-photosynthetic tissues found that all of the MeFtsZ genes had higher expression levels in photosynthetic tissues, especially in younger leaves, and lower expression levels in the non-photosynthetic tissues. During cassava storage root development, the expressions of MeFtsZ2-1 and MeFtsZ2-2 were comparatively higher than MeFtsZ1. The transformed Arabidopsis of MeFtsZ2-1 and MeFtsZ2-2 contained abnormally shape, fewer number, and larger volume chloroplasts. Phytohormones were involved in regulating the expressions of MeFtsZ genes. Therefore, we deduced that all of the MeFtsZs play an important role in chloroplast division, and that MeFtsZ2 (2-1, 2-2) might be involved in amyloplast division and regulated by phytohormones during cassava storage root development.