Elucidation of the regulatory mechanism of SWEET11 phloem parenchyma cell-specific expression

Zhang, Chen

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https://hdl.handle.net/2142/121259 Copy

Description

Title

Elucidation of the regulatory mechanism of SWEET11 phloem parenchyma cell-specific expression

Author(s)

Zhang, Chen

Issue Date

2023-07-12

Director of Research (if dissertation) or Advisor (if thesis)

Chen, Li-Qing

Doctoral Committee Chair(s)

Leakey, Andrew

Committee Member(s)

• Marshall-Colon, Amy
• Hong, Jin

Department of Study

Plant Biology

Discipline

Plant Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• coevolution
• phloem parenchyma
• post-transcriptional regulation
• specific expression
• SWEET transporter
• vascular plants.

Abstract

Phloem transport of photoassimilates from source tissues (mainly mature leaves) to sink tissue (such as roots, fruits, and seeds) is a crucial process for plant growth, development, and reproduction. Phloem loading is the first step of this transport, in which photoassimilates enter the phloem of vascular tissues. Two primary phloem loading strategies exist: apoplasmic and symplasmic loading. Most herbaceous plants utilize an apoplasmic loading strategy, in which sucrose is exported from phloem parenchyma (PP) cells into the apoplasmic cell-wall space through uniporter SWEETs (sugars will eventually be exported transporters). The exported sucrose is then imported into the sieve-element/companion cells (SE/CC) complex via the proton-sucrose symporter (SUT). In Arabidopsis, SWEET11 and SWEET12, exclusively expressed in PP cells of leaves, mediate sucrose efflux from PP to the apoplasm, initiating the first stage of apoplasmic phloem loading. Tight control of sugar flux throughout different cell types is critical for development. Loss-of-function mutants in the AtSWEET11 and AtSWEET12 genes, as well as overexpression of either one under the constitutive 35S promoter, have been found to result in a stunted phenotype, indicating the SWEET11/12 PP-specific expression is vital for sugar flux. However, the mechanism controlling their PP-specific expression is still elusive. In Chapter II, I explored the region of SWEET11/12 that determines the PP-specific expression. The SWEET11/12 promoter region is sufficient to activate reporter genes in PP cells but is constitutively expressed in all leaf cell types. Translational fusion of the SWEET11/SWEET12 promoter with its genomic DNA (gDNA) showed PP specificity. It is necessary to investigate the contribution of the gDNA to the SWEET PP-specific expression. Through truncation analysis, I identified that both the promoter and coding regions of SWEET11/SWEET12 are required for PP-specific expression, while introns are not. Further studies revealed that either one of the TM3 or TM7 duplicated domains within the SWEET11 coding region is sufficient to control SWEET11 PP-specific expression. SWEET11 3′ UTR is essential for the SWEET11 expression pattern in newly emerged leaves. Gene regulation is a highly dynamic and tightly controlled process. Identification of gene expression regulators helps us better understand the mechanism of gene specificity. In Chapter III, I conducted forward and reverse genetic screening to explore the regulation mechanism determining SWEET11 PP specificity. Reverse genetic screening is used to identify regulators and regulation mechanisms for determining SWEET11 PP specificity based on modifying SWEET11. Truncation analysis of the SWEET11 promoter including 5′ UTR narrows down it to a 126 bp region which conveys SWEET11 PP-specific expression. I further explored TM7-mediated PP specificity by modifying its sequence. I excluded transcriptional, translational, and post-translational regulation and concluded that post-transcriptional regulation controls TM7 PP-specific expression. RNA-binding proteins (RBPs) could regulate TM7 PP specificity. To identifiey the regulators contributing to SWEET11 PP-specific expression, I employed EMS mutagenesis on the pSWEET11:SWEET11-GUS:3′ UTR background and screened for the EMS mutants with altered tissue-specific expression patterns. Through this approach, I identified several mutants with altered SWEET11 expression patterns, which will serve as candidate materials for further investigation. SWEETs are found in all plant domains, including bacteria and eukaryotes. Given the critical role of the coding region of SWEET11 in determining its PP-specific expression pattern, I aimed to investigate whether the mechanism responsible for SWEET11 PP-specific expression co-evolved with the evolution of vascular plants. In Chapter IV, I found that clade III SWEETs (sucrose transporting SWEETs) evolved with the vascular plants. I also found that SWEET 8PP-specific expression appeared after the emergence of vascular plants and is independent of the substrate specificity of SWEET11 during evolution. Additionally, I observed that the pSWEET11:ZmSWEET13a-GUS transgenic Arabidopsis only displayed a GUS signal in the PP cells, not the bundle sheath (BS), as seen in maize. This expression pattern suggests that the specific expression of C4 maize SWEET13 may be determined by the coordinated action of the promoter and genomic region or by unique C4-specific regulators. This Ph.D. thesis provides novel insights into the regulatory mechanisms underlying SWEET11 PP-specific expression. The research findings expand our understanding of PP specificity in plants and the evolution of clade III SWEET transporters, making substantial contributions to future studies on PP physiology and development and crop yield improvement.

Graduation Semester

2023-08

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/121259

Copyright and License Information

Copyright 2023 Chen Zhang

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