Expression and functional analyses of EXO70 genes in Arabidopsis implicate their roles in regulating cell type-specific exocytosis

During exocytosis, Golgi-derived vesicles are tethered to the target plasma membrane by a conserved octameric complex called the exocyst. In contrast to a single gene in yeast and most animals, plants have greatly increased number of EXO70 genes in their genomes, with functions very much unknown. Reverse transcription-polymerase chain reactions were performed on all 23 EXO70 genes in Arabidopsis (Arabidopsis thaliana) to examine their expression at the organ level. Cell-level expression analyses were performed using transgenic plants carrying β-glucuronidase reporter constructs, showing that EXO70 genes are primarily expressed in potential exocytosis-active cells such as tip-growing and elongating cells, developing xylem elements, and guard cells, whereas no expression was observed in cells of mature organs such as well-developed leaves, stems, sepals, and petals. Six EXO70 genes are expressed in distinct but partially overlapping stages during microspore development and pollen germination. A mutation in one of these genes, EXO70C1 (At5g13150), led to retarded pollen tube growth and compromised male transmission. This study implies that multiplications of EXO70 genes may allow plants to acquire cell type- and/or cargo-specific regulatory machinery for exocytosis.


Supplementary Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Phylogenetic analysis of EXO70 proteins in different organisms Supplemental Figure S2. Homology matrix tree of 23 EXO70 protein sequences. Supplemental Figure S3. Alignment of the QR motif located near the C-termini of EXO70s from rice and Arabidopsis Supplemental Figure Table S1. Primers used for the analysis of the expression of EXO70 genes Supplemental Table S2. Primers used for amplifying EXO70 promoters for GUS fusion. The tree was constructed using ClustalX, as described in Materials and Methods.
Three clades (Exo70.1, Exo70.2, Exo70.3) , 9 floral plant-conserved clusters (A to I) and one monocot-specific cluster Fx were marked at the right. Four sets of Exo70 proteins identified from completely sequenced genomes of Arabidopsis, rice, sorghum and poplar were used for this analyses. Within the 9 clusters, dicot-and monocot-specific Exo70 proteins were marked in blue and red boxes, respectively.

Supplemental Figure S12. Expression pattern of EXO70D2
A, the AVT expression profile of this member; B, a 3-day-old seedling, the GUS signal was found in root tip, hypocotyls and cotyledons; C, a 7 day-old seedling, GUS expression was observed in hypocotyls, cotyledon and leaf. In addition, a weak signal was localized at root tip; D, the GUS expression was detected in lateral root cap in 7 day-old seedlings; E, the GUS signal was associated with trichome cells of leaf, especially in newly formed ones; F, the GUS expression was found in node of inflorescence and carpels (inset). Bar=0.5mm in B; Bars=1mm in C,F; Bar=0.1mm in E; Bar=50μm in D.

Supplemental Figure S13. Expression pattern of EXO70D3
A, the AVT expression profile of this member; B, a 3-day-old seedling, showing GUS signal in the root tip, and weak signal in the hypocotyl; C, a 7 day-old seedling, showing GUS signal in leaves; D, a root tip of a 7 day-old seedling, GUS expression was located in lateral root cap; E, a lateral root tip of a 7-day old seedling, GUS signal was detected in the lateral root cap ; F, a 7-day old seedling, the GUS expression was observed in developing trichomes on the leaf surface; G, the GUS signal was not found in inflorescences. Bars=0.5mm in B, F; Bars=1mm in C, G; Bars=50μm in D, E.

Supplemental Figure S14. Expression pattern of EXO70E1
A, AVT expression profile of this member; B, 3-day-old seedling, GUS signal was found in cotyledon and root hair region in root; C, 7 day-old seedling, showing strong signal in cotyledon ,leaf and root excluding root tip; D, an enlarged view of root tip, GUS expression was started from elongation region and restricted to root hair cell in root tip. In the other part of root (near hypocotyl), the GUS was distributed in all cell types; E, the GUS expression of root hair cells showed strongest signal in the cells undergoing hair initiation; F, GUS staining was not found in inflorescence. Bar=0.5mm in B; Bars=1mm in C,F; Bar=50μm in D; Bar=0.25 μm in E.

Supplemental Figure S15. Expression pattern of EXO70E2
A, AVT expression profile of EXO70E2 (At5g61010) ; B, 3-day-old seedling, the GUS signal was found in hypocotyl and root hair region; C, 7 day-old seedling, the GUS signal was found strongly expressed in root, leaf and hinge of leaves; D, the inflorescence, showing GUS staining in node of inflorescence.
Bars=0.25mm in B, C; Bar=0.5mm in D.

Supplemental Figure S16. Expression pattern of EXO70F1
A, AVT expression profile of EXO70F1; B, a 3-day-old seedling, GUS signal was found in the root close to hypocotyl; C, a 7 day-old seedling, the signal was found in root and

Supplemental Figure S19. Expression pattern of EXO70H1
A, AVT expression profile of EXO70H1; B, a 3-day-old seedling, obvious GUS signal was found in root except root tip; C, a 7 day-old seedling, the signal was found in similar patterns as 3-day-old seedling, but the expression restricted to root hair region where the root hair undergoing growth; D, an enlarged view of the root tip in B, GUS signal started from elongation zone; E, an enlarged view of root hair region in B, the growing root hair cells was strongly stained; F, Inflorescence, weak GUS signal was located in