using-phylogenetic-analysis-to-investigate-eukaryotic-gene-origin

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Nanjing Agricultural University

The plasma membrane (PM) proton pump ATPases (H(+)-ATPases) are involved in almost all aspects of biology. They are plant specific and several members of this family are supposed to play a key role in nutrient acquisition. At present, only some members of this gene family in plants have been characterized. However, no nutrient uptake associated H(+)-ATPase gene in rice has been functionally analysed. It is reported here that OsA8, a typical PM H(+)-ATPases gene that was predominantly expressed in roots of rice, is down-regulated by nutrient deficiency. The Osa8 mutant had a relatively smaller size and root to shoot biomass ratio, but higher ATPase activity than its wild-type counterparts under phosphorus (P) starvation conditions. Knockout of OsA8 affected the expression of several OsA genes and the high affinity phosphate transporter, OsPT6, and resulted in a higher P concentration in the roots and a lower amount of P in the shoots. These analyses demonstrate that OsA8 not only influences the uptake of P by roots, but also the translocation of P from the roots to the shoots in rice.

Proton pump OsA8 is linked to phosphorus uptake and translocation in rice.

DREB proteins are involved mainly in plant responses to abiotic stresses such as cold, drought or high salinity as well as ABA signalling. However, the function of most rice DREB genes and the underlying molecular mechanisms controlling these responses remains elusive. In this study, ARAG1, a rice DREB gene, was functionally analysed.

ARAG1, an ABA-responsive DREB gene, plays a role in seed germination and drought tolerance of rice.

Jerusalem artichokes (Helianthus tuberosus L.) can tolerate relatively higher salinity, drought and heat stress. In this paper, we report the cloning of a Salt Overly Sensitive 1 (SOS1) gene encoding a plasma membrane Na(+)/H(+) antiporter from a highly salt-tolerant genotype of H. tuberosus, NY1, named HtSOS1 and characterization of its function in yeast and rice. The amino acid sequence of HtSOS1 showed 83.4% identity with the previously isolated SOS1 gene from the Chrysanthemum crassum. The mRNA level in the leaves of H. tuberosus was significantly up-regulated by presence of high concentrations of NaCl. Localization analysis using rice protoplast expression showed that the protein encoded by HtSOS1 was located in the plasma membrane. HtSOS1 partially suppressed the salt sensitive phenotypes of a salt sensitive yeast strain. In comparison with wild type (Oryza sativa L., ssp. Japonica. cv. Nipponbare), the transgenic rice expressed with HtSOS1 could exclude more Na(+) and accumulate more K(+). Expression of HtSOS1 decreased Na(+) content much larger in the shoot than in the roots, resulting in more water content in the transgenic rice than WT. These data suggested that HtSOS1 may be useful in transgenic approaches to improving the salinity tolerance of glycophyte.

Functional analyses of a putative plasma membrane Na+/H+ antiporter gene isolated from salt tolerant Helianthus tuberosus.

In plants, KT/HAK/KUP is the largest potassium (K) transporter family, however, few of the members have had their physiological functions characterized in planta. Here we studied OsHAK5 of the KT/HAK/KUP family in rice. We determined its cellular and tissue localization, and analyzed its functions in rice using both OsHAK5 knockout mutants and overexpression lines at three genetic backgrounds. GUS reporter driven by OsHAK5 native promoter indicated OsHAK5 expression in various tissue organs from root to seed, abundantly in root epidermis and stele, the vascular tissues and mesophyll cells. Net K influx rate in roots and K transport from roots to aerial parts was severely impaired by OsHAK5 knockout but increased by OsHAK5 overexpression in 0.1 and 0.3 mM K external solution. The contribution of OsHAK5 to K mobilization within the rice plant was confirmed further by the change of K concentration in the xylem sap and K distribution in the transgenic lines when K was removed completely from the external solution. Overexpression of OsHAK5 increased [K]/[Na] ratio in the shoots and salt stress tolerance (shoot growth), while knockout of OsHAK5 decreased [K]/[Na] ratio in the shoots resulting in sensitivity to salt stress. Taken together, these results demonstrate that OsHAK5 plays a major role in K acquisition by roots faced with low external K and in K upward transport from roots to shoots in K deficient rice plants.

The role of OsHAK5 in potassium acquisition and transport from roots to shoots in rice at low potassium supply levels.

Potassium (K) absorption and translocation in plants rely upon multiple K transporters for adapting varied K supply and saline conditions. Here, we report the expression patterns and physiological roles of OsHAK1, a member belonging to the KT/KUP/HAK gene family in rice (Oryza sativa L.). The expression of OsHAK1 is up-regulated by K deficiency or salt stress in various tissues, particularly in the root and shoot apical meristem, the epidermises and steles of root, and vascular bundles of shoot. Both oshak1 knockout mutants in comparison to their respective Dongjin or Manan wild types showed a dramatic reduction in K concentration and stunted root and shoot growth. Knockout of OsHAK1 reduced the K absorption rate of unit root surface area by ∼50-55 and ∼30%, and total K uptake by ∼80 and ∼65% at 0.05-0.1 and 1 mm K supply level, respectively. The root net high-affinity K uptake of oshak1 mutants was sensitive to salt stress but not to ammonium supply. Overexpression of OsHAK1 in rice increased K uptake and K/Na ratio. The positive relationship between K concentration and shoot biomass in the mutants suggests that OsHAK1 plays an essential role in K-mediated rice growth and salt tolerance over low and high K concentration ranges.

Rice potassium transporter OsHAK1 is essential for maintaining potassium-mediated growth and functions in salt tolerance over low and high potassium concentration ranges.

SWEETs represent a new class of sugar transporters first described in plants, animals, and humans and later in prokaryotes. Plant SWEETs play key roles in phloem loading, seed filling, and nectar secretion, whereas the role of archaeal, bacterial, and animal transporters remains elusive. Structural analyses show that eukaryotic SWEETs are composed of 2 triple-helix bundles (THBs) fused via an inversion linker helix, whereas prokaryotic SemiSWEETs contain only a single THB and require homodimerization to form transport pores. This study indicates that SWEETs retained sugar transport activity in all kingdoms of life, and that SemiSWEETs are likely their ancestral units. Fusion of oligomeric subunits into single polypeptides during evolution of eukaryotes is commonly found for transporters. Phylogenetic analyses indicate that THBs of eukaryotic SWEETs may not have evolved by tandem duplication of an open reading frame, but rather originated by fusion between an archaeal and a bacterial SemiSWEET, which potentially explains the asymmetry of eukaryotic SWEETs. Moreover, despite the ancient ancestry, SWEETs had not been identified in fungi or oomycetes. Here, we report the identification of SWEETs in oomycetes as well as SWEETs and a potential SemiSWEET in primitive fungi. BdSWEET1 and BdSWEET2 from Batrachochytrium dendrobatidis, a nonhyphal zoosporic fungus that causes global decline in amphibians, showed glucose and fructose transport activities.-Hu, Y.-B., Sosso, D., Qu, X.-Q., Chen, L.-Q., Ma, L., Chermak, D., Zhang, D.-C., Frommer, W. B. Phylogenetic evidence for a fusion of archaeal and bacterial SemiSWEETs to form eukaryotic SWEETs and identification of SWEET hexose transporters in the amphibian chytrid pathogen Batrachochytrium dendrobatidis.

Phylogenetic evidence for a fusion of archaeal and bacterial SemiSWEETs to form eukaryotic SWEETs and identification of SWEET hexose transporters in the amphibian chytrid pathogen Batrachochytrium dendrobatidis.

This study investigated the role of the sugar transporter OsSWEET11 during the early stage of rice caryopsis development using β-glucoronidase (GUS) to represent its expression, together with clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9)-mediated knockout, cross-fertilization and RNA sequencing (RNA-seq) analyses. The results showed that OsSWEET11 was expressed strongly in developing caryopsis, particularly in the ovular vascular trace, nucellar epidermis and cross cells. The knockout of OsSWEET11 significantly decreased the sucrose concentration in the mutant embryo sacs and led to defective grain filling compared with that of the wild-type (WT) plant. Moreover, the expression of 2,549 genes in the mutant caryopsis was affected. The grain weight and seed setting percentage were also decreased in the mutants. The cross-fertilization of the mutant and WT rice revealed that the mutated maternal donor induced defective grain filling. These results strongly suggested that OsSWEET11 played an important role in sucrose release from maternal tissue to the maternal-filial interface during the early stage of caryopsis development. It might also induce sucrose release from the ovular vascular trace and cross cells of developing caryopsis. These findings bridge the gap in the understanding of post-phloem sugar transport during the early stage of rice caryopsis development.

Yibing Hu

College of Resources & Environmental Sciences

Nanjing Agricultural University

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Yibing Hu

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Nanjing Agricultural University

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

College of Resources & Environmental Sciences