The rice ASR5 protein

Abstract

Under acidic soil conditions, aluminum (Al) becomes available to plants, which must cope with its toxicity by mechanisms involving both internal and external detoxification. Rice is the most Al-tolerant among the crop species, with Al detoxification being managed by both mechanisms. Recently, we focused on ASR (Abscisic acid, Stress and Ripening) gene expression analyses and observed increased ASR5 transcript levels in roots and shoots in response to Al. In addition, ASR5 RNAi knock down plants presented an Al-sensitive phenotype. A proteomic approach showed that ASR5 silencing affected several proteins related to photosynthesis in RNAi rice shoots. Furthermore, an ASR5-GFP fusion in rice protoplasts revealed for the first time a chloroplast localization of this protein. Because it is well known that Al induces photosynthetic dysfunction, here we discuss the hypothesis that ASR5 might be sequestered in the chloroplasts as an inactive transcription factor that could be released to the nucleus in response to Al to regulate genes related to photosynthesis.

According to the FAO, aluminum (Al) toxicity is the most serious problem in the soil constraints on agriculture, second to erosion hazards.¹ Al is a very abundant metal in the earth’s crust, but it becomes a problem under acidic conditions under which Al becomes soluble and is taken up by plants. Plants have developed strategies to cope with Al via internal and external detoxification systems. The release of organic acids that form complexes and prevent Al entry into cells has been well documented.^2-4 However, some plants have managed to mediate Al by chelation and storage in the vacuoles.^5,6 Among crops, rice is the most Al-tolerant species due to its capacity to cope with Al using both mechanisms.^7,8 Although rice can exclude Al from the roots, a portion of the Al can enter the cells and be transported to the shoots.¹⁰

In recent years, different studies aiming to decipher Al-tolerance in rice permitted the identification of a few genes,⁹ and our previous work has added a new association between ASR genes (Abscisic acid, Stress and Ripening) and Al tolerance.¹¹ The ASR genes are also involved in many abiotic and biotic stresses (for a review, see 12). The exact function, however, remains enigmatic as the possible roles of the ASR genes cannot be deduced by sequence homology with other known proteins.¹² Most of the ASR proteins reported to date are located in the nucleus and possess DNA-binding activities. ASR from grape, for example, was able to bind to the promoter of a hexose transporter gene and regulate its expression.¹³ Selex-binding experiments showed that ASR1 from tomato exhibit DNA sequence-preferential binding.¹⁴ Furthermore, ASR1 from tomato was able to compete for the transcription factor ABI4 binding motif when overexpressed in Arabidopsis.¹⁵

We have previously shown that ASR5 transcripts are increased in response to Al in roots and shoots.¹¹ ASR5_RNAi plants presented a high Al sensitivity and a trichome-less phenotype. In addition, a proteomic approach revealed several proteins of which the expression was affected in shoots due to the silencing of ASR5. Furthermore, we confirmed the nuclear localization of ASR5 using rice calli transformed with an ASR5_GFP fusion construct. However, when this construct was transformed in protoplasts, ASR5 was accumulated in the chloroplast precursors (etioplastids) of rice leaves protoplasts grown under dark conditions (Fig. 1A) and chloroplasts (Fig. 1B) of rice leaves protoplasts grown under light conditions. GFP alone was used as positive control (Fig. 1C). This new and intriguing localization for ASR5 led us to speculate that ASR5 plays a role in this organelle, most likely linking the signaling between the chloroplasts and nucleus and regulating the expression of chloroplast proteins. Al can enter rice chloroplasts¹⁶ and decrease the photosynthetic ratio.^17,18 The ASR5 silencing affected the expression of at least 41 proteins in rice leaves.¹¹ A total of 19 out of these 41 proteins contains the chloroplast transfer peptide (cTP) signal, as predicted by sequence analyses^19,20 and, were found to be differentially expressed in our RNAi line. The identified proteins are involved in such processes as photosynthesis, electron transport and stress responses (Table 1). The overexpression of ASR5 (named ASR1 in the cited report) in rice, increased the tolerance to cold, and the plants presented approximately 2-fold higher Fv/Fm values for the photosynthetic efficiency when compared with non-transformed plants.²¹ Out of the 19 proteins with a cTP signal found in our plants, 14 were decreased in the ASR5_RNAi line and might be helpful to explain the Al sensitivity of the RNAi plant. These genes may be regulated by ASR5 in the nucleus because most of the proteins with cTP signals are encoded in the nuclear genome, translated in the cytosol and post-translationally imported into the chloroplasts.²²

Figure 1. Rice protoplast transformation with ASR5-GFP fusion. From upper to lower – The ref fluorescence of chlorophyll, ASR5-YFP fluorescence and merged images using a confocal laser scanning microscope. (a) Protoplast from rice leaves grown under dark conditions. (b) Protoplast from rice leaves grown under light conditions. (c) Vector with GFP only used as positive control. No green fluorescence was detected in negative control (data not shown). Bar = 10µM.

Table 1. Proteins differentially expressed in RNAi_ASR rice plants.

Function	Expression	Protein name	Accession (NCBI)
Protein degradation
1	↓	Heat shock protein 70	gi|222631026
2	↓	Mitochondrial chaperonin	gi|115488160
Photosynthesis
3	↓	ATP synthase gamma chain	gi|115472339
4	↑	Putative oxygen-evolving enhancer protein	gi|115436780
5	↓	Ferredoxin-NADP(H) oxidoreductase	gi|41052915
6	↑	PSI reaction center subunit IV	gi|34394725
Carbohydrate metabolism
7	↓	Putative transketolase	gi|28190676
8	↓	Sedoheptulose 1–7 bisphosphatase	gi|27804768
9	↓	Putative uridylyltransferase-related	gi|187608845
10	↓	Ribulose-5-phosphate-3-epimerase	gi|4105561
Cellular component
11	↓	Fibrillin-like protein	gi|29367475
12	↓	PAP fibrillin family domain protein	gi|115486133
Amino acid metabolism
13	↓	Putative glycine cleavage system H protein	gi|115482934
Stress response
14	↑	Class III peroxidase (OsPrx111)	gi|20286
15	↓	Chloroplastic lipocalin (Os04 g0626400)	gi|115460690
16	↑	Putative superoxide dismutase [Cu-Zn]	gi|42408425
Transport of electrons
17	↓	Oxidoreductase NAD-binding domain	gi|115445869
18	↑	2Fe-2S iron-sulfur cluster binding domain	gi|18698985
Nucleotide binding
19	↓	Chloroplast elongation factor Tu	gi|218191089

The proteins differentially expressed in transgenic rice shoots silenced for ASR5 with a chloroplast transfer peptide signal (cTP), as predicted by PCLR and ChloroP 1.1 software.

Our hypothesis is that ASR5 is stored inside the chloroplast until specific conditions (such as Al stress) require their activity in the nucleus; a scenario proposed for certain other dual-target transcription factors.²³ For example, Whirly1 is localized in both the nucleus and plastids of the same cells in barley leaves.²⁴

Curiously, the ASR5 N-terminal region lacks the expected cTP sequence required to target this protein to the chloroplast. Notwithstanding, in Arabidopsis chloroplast preparations, a total of 604 proteins encoded in the nuclear genome were found, and surprisingly, only 376 were predicted to have the cTP when analyzed by a chloroplast-targeting prediction tool.²⁵ Proteins with cTP signals are targeted to the chloroplast surface and imported across the double-membrane envelope by translocons in the outer and inner envelope membranes, termed TOC and TIC, respectively.²⁶ However, increasing evidence shows that some proteins can enter the chloroplast by a TOC-TIC independent pathway.^27-29 The transcriptional responses to abrupt environmental changes often have to occur rapidly and might require a release of pre-produced inactive transcription factors. A controlled sequestering of proteins at intracellular membranes seems to be an established way of controlling gene expression and intracellular communication.²³ Furthermore, a potential myristoylation site in rice ASR5 sequence is conserved among several ASR proteins at the same location when aligned.³⁰ The myristoyl residue may bind to a hydrophobic pocket and thus confer structural stability to a protein.³¹ The most obvious function of the myristoyl moieties is to mediate membrane binding³² and is frequently found in proteins shuttling across membranes, where the myristoyl residue inserts into the inner lipid layer of the plasma membrane and facilitates membrane interaction.³⁰

In tobacco, Al accumulated in the chloroplasts after entering the cells, reacted with or replaced the non-heme iron between the QA and QB binding sites and blocked PSII electron transport, resulting in PSII photochemical damage and the inhibition of photosynthesis.³³ Thus, Al could be the signal for ASR5 release from the chloroplasts to regulate photosynthesis related gene expression and cope with Al toxicity in rice shoots.

Materials and Methods

The complete sequence of ASR5 was amplified using cDNA library of rice Japonica cultivar, sequenced and cloned fusionated with YFP coding sequence at its N-terminus into the Gateway vector pART7-YFP.³⁴ Protoplast isolation was performed according to Chen et al. (2006)³⁵ and protoplast transformation according to Tao et al. (2002).³⁶ Transformed rice protoplasts grown under dark and under light conditions were incubated in the dark for 24–48 h at 27°C prior to imaging. Fluorescence microscopy was performed with an Olympus FluoView 1000 confocal laser-scanning microscope equipped with a set of filters capable of distinguishing green and yellow fluorescent proteins (EGFP and EYFP, respectively) and plastid autofluorescence. Images were captured with a high-sensitivity photomultiplier tube detector. A vector containing only GFP was used as positive control and an empty vector was used as a negative control. The experiment was performed in biological triplicate.