Functional Analysis Of The Sugar Transporters SWEETs And SUT4 In Populus

In higher plants, sugars function as the main energy reserves, important signaling molecules, osmotic adjustment substances and the building blocks for other organic compounds which play important roles during the entire life cycle. Sugar transporters play crucial roles in many steps of sugar transport, including long-distance distribution of sugars through vascular tissues cell-to-cell and within cells. Therefore, they are important in plant growth and development. Now, althrough some sugar transporters have been partially described in Arabidopsis and rice, the function of sugar transporters in woody plant poplars has not been systematically investigated. Compared with herbaceous annuals, woody plants have many specific developmental processes. In trees, wood formation needs carbon import from the photosynthetic tissues. Therefore, research on the function of the sugar transporters in Populus contribute to reveal the allocation mechanism of trees, and provide fundamental basis for regulation of wood growth by regulation carbon allocation. In this study, a comprehensive identification and characterization of the SWEET family in Populus was performed, including evolutionary relationship, expression profiling, and sugar transport properties analyses. The potential roles of PtSWEET5, PtSWEET7 and PtSWEET17 a in growth were characterized in transgenic poplars. In addition, we further investigated the roles of PtSUT4 of the SUT gene family in secondary growth. The main results are as follows:1. This study characterized 27 SWEET genes in Populus, which is roughly 1.59-fold more than the number of the SWEET gene in Arabidopsis. Phylogenetic analysis showed that Populus SWEET members could be grouped into four clade, the exon/intron organization and motif composition were relatively conserved in each clade. Segmental duplications and tandem duplication events contributed to the expansion of the PtSWEET gene family.2. To explore the subcellular localizations of the PtSWEETs, 9 selected PtSWEETs,representing each clade were examined by fusing them in-frame to the N-terminus of YFP andtransiently expressing them in N. benthamiana leaf epidermal cells. Confocal microscopy showed that PtSWEET4-YFP, PtSWEET7-YFP and PtSWEET15b-YFP were targeted to the plasma membrane. The YFP signals of the PtSWEET17b-YFP and PtSWEET17d-YFP were detected at the tonoplast. PtSWEET2c-YFP, PtSWEET3b-YFP, and PtSWEET17a-YFP signals were observed primarily in the ER, and PtSWEET16a-YFP was in the Golgi. These results implied that these PtSWEETs might play important roles in sugar transportation and allocation cell-to-cell or intracellular. To investigate the transport function of the PtSWEETs, 12 selected members representing each clade were expressed separately in the hexose absorb defect S.cerevisiae strain EBY.VW4000. Complementation growth assays showed that PtSWEET4, 5and 7could transport glucose, mannose and galactose in yeast cells, PtSWEET15 a could transport galactose. These suggested PtSWEETs might have the ability to transport a variety of sugar.3. Real-time quantitative PCR analysis showed that PtSWEETs have distinct expression patterns in various tissues. PtSWEET1 a and PtSWEET1 b were expressed mainly in the roots;PtSWEET2c、3b and 10 a were expressed mainly in mature leaves; PtSWEET5、7、10b and 10 c were expressed mainly in the phloem; PtSWEET15 b was expressed specifically in the xylem;and PtSWEET4 、 5 and 15 a were expressed mainly in the male flowers; PtSWEET9 and PtSWEET11 were exclusively expressed in male and female flowers. Histochemical GUS assays showed that PtSWEET2 C exhibited a higher expression level in mature leaves than in other tissues; PtSWEET7 was specifically expressed in the phloem; PtSWEET17 a exhibited a high expression level in the phloem, xylem, ray cells, and roots; PtSWEET15 b was specifically expressed in the xylem. These were in line with the quantitative PCR analysis. The diversified expression patterns of the PtSWEETs suggested that they may participate in a diverse array of biological processes in Populus, and the members were highly expressed in stem may involve in the sugar transport in the process of xylem development.4. To study the function of PtSWEET5, PtSWEET7 and PtSWEET17 a, the overexpression lines of PtSWEET5, PtSWEET7 and PtSWEET17 a were generated separately. Overexpression of PtSWEET5 facilitated xylem and phloem development. PtSWEET5-overexpressing poplarlines displayed the phenotypes of growth retardation and precocious senescence in root and leaf. FDA/PI double staining analysis showed that 35S: PtSWEET5 transgenic poplar lines displayed reduced cell viability in the roots. In contrast, Phenotypic and histological section analyses showed that transgenic poplars overexpressing PtSWEET7 and PtSWEET17 a exhibited increased stem growth and enhanced xylem development, suggesting roles in the sugar allocation to the woody tissues.5. Subcellular localization and qRT-PCR analysis showed that Pt SUT4 gene encoding a tonoplast-localized sucrose transporter. The PtSUT4 gene were ubiquitously expressed and showed high expression level in mature leaves, lignified stem, phloem, and flowers. The transgenic lines(S1 and S12) showed 22% and 17% increase in stem height, 9% and 7%increase in ground diameter, respectively. In addition, the two transgenic lines showed 32%and 21% increase in the xylem width of the seventh internodes. The PtSUT4 overexpression lines(S1 and S12) exhibited 24% and 21% higher photosynthetic rate compared with the non-transgenic controls, respectively. Overexpression of PtSUT4 may have positive feedback effect on the photosynthesis through the enhancement of sucrose loading in source tissues and the unloading in sink tissues. This resulted in the promoted both high growth and radial growth(xylem development) in transgenic poplar plants.This study identified 27 PtSWEET genes and provided insights into their evolution and certain sugar transport features of these genes. The differentiation and diversification of expression patterns, the different localizations, and different sugar transport properties compared with AtSWEET gene family implied that they participate in multiple developmental processes in woody species. Indeed, Pt SWEET5 might be involved in senescing process of Populus, PtSWEET7, PtSWEET17 a and PtSUT4 play essential roles in secondary growth.These data serve as a basis for further studies on the roles and regulation mechanism of sugar transporters in woody plants. Moreover, these provided theoretical basis for accelerated growth of trees and improvement of wood properties of the trees.