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At14a-Like1 participates in membrane-associated mechanisms promoting growth during drought in Arabidopsis thaliana
Thursday, 2015/08/20 | 08:20:21

M. Nagaraj Kumar, Yi-Fang Hsieh, and Paul E. Verslues

 

Significance

Drought is a major cause of lost agricultural productivity. Even moderate water limitation can lead to down-regulation of plant growth; however, the underlying mechanisms of stress sensing and growth regulation are little understood. We identified At14a-Like1 (AFL1) and its interacting proteins protein disulfide isomerase 5 (PDI5) and NAI2 as positive and negative regulators, respectively, of growth and proline accumulation. Despite numerous ideas that membrane-based mechanisms are important for drought sensing and initial signaling, AFL1 is one of only a few membrane proteins with a demonstrated effect on drought resistance. AFL1 structure, localization, and interaction with endomembrane proteins indicate novel functions in drought signaling. Increased growth of AFL1 overexpression in plants under stress without negative effects on unstressed plants make AFL1 an attractive target for biotechnology.

 

Abstract

Limited knowledge of how plants regulate their growth and metabolism in response to drought and reduced soil water potential has impeded efforts to improve stress tolerance. Increased expression of the membrane-associated protein At14a-like1 (AFL1) led to increased growth and accumulation of the osmoprotective solute proline without negative effects on unstressed plants. Conversely, inducible RNA-interference suppression of AFL1 decreased growth and proline accumulation during low water potential while having no effect on unstressed plants. AFL1 overexpression lines had reduced expression of many stress-responsive genes, suggesting AFL1 may promote growth in part by suppression of negative regulatory genes. AFL1 interacted with the endomembrane proteins protein disulfide isomerase 5 (PDI5) and NAI2, with the PDI5 interaction being particularly increased by stress. PDI5 and NAI2 are negative regulatory factors, as pdi5, nai2, and pdi5-2nai2-3 mutants had increased growth and proline accumulation at low water potential. AFL1 also interacted with Adaptor protein2-2A (AP2-2A), which is part of a complex that recruits cargo proteins and promotes assembly of clathrin-coated vesicles. AFL1 colocalization with clathrin light chain along the plasma membrane, together with predictions of AFL1 structure, were consistent with a role in vesicle formation or trafficking. Fractionation experiments indicated that AFL1 is a peripheral membrane protein associated with both plasma membrane and endomembranes. These data identify classes of proteins (AFL1, PDI5, and NAI2) not previously known to be involved in drought signaling. AFL1-predicted structure, protein interactions, and localization all indicate its involvement in previously uncharacterized membrane-associated drought sensing or signaling mechanisms.

 

See: http://www.pnas.org/content/112/33/10545.abstract

PNAS August 18, 2015 vol. 112 no. 33 10545–10550

 

Fig. 1. AFL1 promotes growth and alters the stress transcriptome. (A) Root elongation and dry weight of AFL1 O.E. and RNAi K.D. plants after transfer to unstressed control media (−0.25 MPa) or two low ψw stress severities (−0.7 and −1.2 MPa). Data are means ± SE (n = 12 from two independent experiments). EV, empty vector control for the K.D. lines; DEX, dexamethasone; WT, wild-type (Columbia-0). Significant differences compared with WT are indicated by * (P ≤ 0.05). (B) Representative seedlings of WT and AFL1 O.E. 10 d after transfer to the respective treatments. (Scale bars, 1 cm.) (C) Rosette fresh weight and dry weight of AFL1 O.E. plants relative to WT in well-watered or controlled soil drying treatments. Data are means ± SE (n = 10–12 from three independent experiments). Significant differences compared with WT are indicated by * (P ≤ 0.05). (D) Proline accumulation of AFL1 O.E. and K.D. lines across a range of low ψw severities (means ± SE, n = 12–36, combined from two independent experiments; significant differences marked by *). (E) Microarray gene expression profiles of WT and AFL1 O.E. (35S:YFP-AFL1) analyzed to determine whether increased AFL1 enhanced or antagonized the WT transcriptional response to low ψw. Full microarray results are shown in Dataset S1. (F) Quantitative RT-PCR analysis of RD21. Data are means ± SE (n = 4) from two independent experiments. Significant differences (P ≤ 0.05) relative to WT are indicated (*).

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