Plant annexins and their involvement in stress responses

Accepted Manuscript Title: Plant annexins and their involvement in stress responses Authors: Deepanker Yadav, Prasanna Boyidi, Israr Ahmed, Puluguratha Bharadwaja Kirti PII: DOI: Reference:

S0098-8472(18)30289-2 https://doi.org/10.1016/j.envexpbot.2018.07.002 EEB 3498

To appear in:

Environmental and Experimental Botany

Received date: Revised date: Accepted date:

22-2-2018 10-6-2018 4-7-2018

Please cite this article as: Yadav D, Boyidi P, Ahmed I, Kirti PB, Plant annexins and their involvement in stress responses, Environmental and Experimental Botany (2018), https://doi.org/10.1016/j.envexpbot.2018.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Plant annexins and their involvement in stress responses Deepanker Yadav* , Prasanna Boyidi, Israr Ahmed, Puluguratha Bharadwaja Kirti* Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India † Present addresse: Department of Fruit Tree Sciences, Institute of Plant Sciences,

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Agricultural Research Organization (ARO), Volcani Center, Israel, *

Correspondence should be addressed to [email protected] (Deepanker Yadav) and

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[email protected] (P B Kirti), Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Prof. C. R. Rao Road, Gachibowli, Hyderabad, 500046, India, Tel:

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0091-40-23134545 / Fax: +914023010120

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Highlights

Annexins are an evolutionarily conserved family of proteins that are known to be involved in important biological processes such as membrane trafficking, cytoskeletal organization, cellular homeostasis and ion transport

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Annexin participation in diverse cellular functions highlight their essential role in plant growth and development, and also their importance in crop improvement programs for enhancing multiple stress tolerance

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An attempt has been made to develop hypothetical sigbal cascades involving annexins in stress tolerance using the published literature

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Abstract

Annexins, which form an evolutionarily conserved family of proteins are known to be

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involved in important biological processes such as membrane trafficking, cytoskeletal organization, cellular homeostasis and ion transport. They are widely known for mediating plant stress responses. Although, the mechanism involved in these responses is not deciphered clearly, several attempts in this direction have strengthened our understanding of the different components involved in annexin-mediated stress responses in plants, which prompted us to link and hypothesize their involvement by predicting a possible relation with different 1

elements participating in stress signaling. In the light of past and present findings, we discuss the structural and cellular properties of plant annexins emphasizing their stress-mediated roles and propose an annexin-mediated signaling cascade in this review, which has not been dealt with earlier. Annexin participation in diverse cellular functions highlight their essential role in plant growth and development and also their importance in crop improvement programs for enhancing multiple stress tolerance.

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Key words Plant annexins; membrane binding; calcium binding; signaling cascade; stress response

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. Introduction

Since the first isolation of an annexin from animal cells as a vesicle fusion protein (Creutz et

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al., 1978), annexins have been the subject of multidisciplinary research for nearly forty years.

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Their identification in other organisms including plants resulted in their expansion to a superfamily of proteins, which have been subgrouped into seven different families viz.,

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ANXA-G (Fernandez et al., 2017). The family ANXA includes vertebrate annexins and

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ANXB comprises invertebrate annexins; annexins from fungi and unicellular eukaryotic organisms were assigned to ANXC. ANXD consists of plant annexins and it comprises 40+

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subfamilies (Clark et al., 2012). The family ANXE includes protist annexins, ANXF represents bacterial annexins and ANXG includes putative archaeal annexins (Fernandez et al.,

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2017; Moss and Morgan, 2004). Separate attempts have also been made to understand the annexin phylogeny in vertebrates (Fernandez et al., 2017), plants (Clark et al., 2012), fungi (Khalaj et al., 2015), protists (Einarsson et al., 2016) and bacteria (Fernandez et al., 2017;

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Kodavali et al., 2014).

Initial research on annexins primarily focused on the vertebrate members and their association with membrane lipids and cytoskeletal proteins that indicated their proposed participation in

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intracellular transport processes leading to vesicle translocation, fusion and transcytosis (Fernandez et al., 2017; Potez et al., 2011; Tebar et al., 2014) . Initially, annexins have been defined as Ca2+ mediated, membrane lipid associated proteins, but the report of

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independent membrane interaction and the presence of other conserved structural motifs, distinct functional domains, unique amino-termini and varied forms of architecture broke this

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paradigm and proposed integrated roles and mechanisms for individual annexin functions (Fernandez et al., 2017). Initially identified members of plants and vertebrate annexins mainly consist of a homologous tetrad of 68 aa ANX domains, which probably originated from a monomeric annexin (Fernandez et al., 2017). Later, many other alternative forms of architecture for the annexin proteins have also been reported, which included 1-20 ANX domains (Crompton et al., 1988;

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Fernandez et al., 2017). The members of ANXD family have <45% amino acid (aa) identity with animal annexins. However, they still preserve the unique annexin fold with the secondary structure assembled into the characteristic tetrad of the four

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homologous domains (Hofmann, 2004; Moss and Morgan, 2004). Each annexin repeat comprises five α- helices (A-E) connected with loops forming a helix-loop-helix structure and the membrane binding sites are situated in the AB and DE loops. Plant annexins also have

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more surface area on the protein because of the presence of extra clefts and grooves when compared to the animal annexins suggesting a wider range of interaction partners for the

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annexin proteins and wider interactions within the cell (Clark et al., 2001; Mortimer et al.,

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2008).

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The Ca2+ binding sites for plant annexins have been confirmed by the crystal structure of a calcium-bound Anx(Gh) (Hu et al., 2008). Subsequently, the Ca2+ binding mechanism of

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annexins was reviewed by Konopka-Postupolska et al. (2011). The most recent attempt to understand plant annexin structure was a phylogenetic comparison study by Clark et al. (2012) in which an alignment of 400 full-length plant annexin protein sequences was subjected to

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statistical evolutionary analysis by HMMER3 (Finn et al., 2011) to develop a pHMM sequence model (Clark et al., 2012). This model defined a ‘typical consensus’ plant annexin,

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which is comparable with an analogous pHMM model proposed earlier for vertebrate annexins (Moss and Morgan, 2004). The model shows a prominent conservation of Ca2+ coordinating sites (GxGT...38 residue..E/D) in the repeat I with a general loss of this binding

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capacity in repeats II/III and a moderate conservation in repeat IV (Clark et al., 2012). The sequestration of Ca2+ ion at the lipid-annexin interface appears to depend on the degree of endonexin conservation and annexin oligomerization (Laohavisit and Davies, 2011). The annexin- membrane interaction is regulated by different factors like Ca2+, pH and the nature of phospholipid head groups on the membrane (Gerke et al., 2005; Gerke and Moss, 2002; Potez et al., 2011). Individual annexin repeats have been shown to have different lipid specificities and are therefore, not equivalent in the membrane-binding process (Laohavisit and Davies, 3

2011). In addition to their association with the plasma membrane, annexins were also found to be associated with different endomembrane structures including tonoplast (Carter et al., 2004; Seals and Randall, 1997), nuclear membrane (de Carvalho Niebel et al., 1998), chloroplast envelope (Seigneurin-Berny et al., 2000) and thylakoid (Friso et al., 2004). Different proteome and transcriptome studies confirmed the ubiquitous presence of annexins in various plant tissues and also their dynamic expression (Table 1). Localization studies

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based on protein fractionation, immunohistochemistry and live cell imaging confirmed the cytosolic abundance and membrane association of annexins, and their localization to various sites likes nucleus, nucleolus and extracellular matrix (Clark et al., 1998; Kovács et al., 1998),

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chloroplast stroma (Rudella et al., 2006) and phloem sap (Giavalisco et al., 2006) has also been reported. Since many of these findings were based on wide scale techniques, there is a need for further specific validation using in vivo studies to establish the subcellular localization and association of plant annexins with the various endomembrane structures.

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Their intra- and extracellular presence and dynamic interaction with Ca2+ and membranes and

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other cellular components (Table 2 and 3) demonstrate their functional diversity. Some of

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their known functions such as ion permeability, vesicle transport, exocytosis, signal sensing

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and signal transduction have been discussed earlier with respect to plant annexins (reviewed by Clark et al., 2012; Davies, 2014; Fernandez et al., 2017; Himschoot et al., 2017; Jami et al., 2012a; Konopka-Postupolska et al., 2011; Laohavisit and Davies, 2011). It was always a

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difficult task to predict an annexin mediated signaling cascade within the plant cell due to the lack of complete data on the interacting partners (Table 2) and the regulatory components.

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However, many new findings pertaining to the identification of various regulatory components of annexin- mediated stress responses and also their involvement in distant signaling prompted

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us to hypothesize annexin mediated signaling mechanism in plant developmental and stress responses.

The present review tries to focus on the annexin mediated signaling mechanism with a special

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emphasis on their importance in stress responses. Further, the possibility of their deployment in the crop improvement program has also been discussed. Annexins in plant signaling Ca2+ and membrane binding property of annexins is pivotal to their involvement in various signaling processes (Alvarez-Martinez et al., 1997; Arpat et al., 2004; Clark et al., 2012; Davies, 2014; Himschoot et al., 2017; Konopka-Postupolska, 2007; Konopka-Postupolska and 4

Clark, 2017; Morel and Gruenberg, 2009; Reddy and Reddy, 2004). Many studies have also reported the Ca2+ transport activity of annexin proteins as an important component in the signaling process (Table 3).

The calcium-based intracellular signaling system is used

ubiquitously to couple extracellular stimuli to their characteristic intracellular responses. Concentration of cytosolic free Ca2+ controls the operation of Ca2+- based signaling in any organism. In the plant cell, resting [Ca2+]cyt is in the range of 100-200 nM (Medvedev, 2005; Wilkins et al., 2016). Stimulation causes its increase by a factor of 10 or 20 (Bose et al.,

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2011). An increase in [Ca2+]cyt is achieved by the controlled entry of Ca2+ from extracellular or intracellular compartments, which possess relatively higher levels of Ca2+ compared to the

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cytosol. The cell contains various proteins for the tight regulation of Ca2+ influx in the cytosol. Annexins also facilitate ion ( Ca2+ , K+ ) conductance across the plasma membrane (Table 3). Moreover, they have the ability to sense the Ca2+ changes as well, which is required for the next step of the Ca2+ based signaling where a suite of proteins whose Ca2+-binding properties

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allow them to sense and respond to this stimulus based increase in [Ca2+]cyt. All these Ca2+

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interacting proteins have certain motifs for Ca2+ interaction (Clark et al., 2012; Edel et al.,

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2017; Fernandez et al., 2017; Marchadier et al., 2016).

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With respect to the channel properties of plant annexins and annexin mediated plant signaling, studies in Arabidopsis thaliana contributed immensely in this direction; different findings suggested annexin integration with the membrane and the capability to form Ca2+ permeable

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channels in planar lipid bilayers (reviewed by Shabala et al., 2015). Annexin 1 of Arabidopsis thaliana is also known to be involved in root epidermal plasma membrane Ca2+- permeable

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conductance (Laohavisit et al., 2012). Under NaCl stress, it has been shown to help in generating ROS-induced Ca2+ influx and upregulation of SOS1- Na+/H+ (Laohavisit et al.,

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2013, Fig 1).

The involvement of ROS as a component of signal transduction has been widely proposed and accepted (reviewed by Shabala et al., 2015). On the other hand, stress tolerance properties

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shown for different proteins favored the hypothesis of the minimization of ROS levels directly or indirectly by these proteins leading to stress tolerance. Besides maintaining below toxic levels of ROS to keep the cell metabolism on its normal track, these antioxidants could play roles in transmitting ROS signaling. Moreover, the variance in the structure and capacity of such redox- active antioxidant components might also contribute to the spatial and temporal specificity to the ROS signaling (Noctor and Foyer, 2016). Additionally, they provide essential information on cellular redox state, and affect the expression of biotic and abiotic 5

stress responsive genes to maximize tolerance/ defense (Foyer and Noctor, 2005). It has also been widely acclaimed that ROS act as signals at only low concentrations and causes damage at higher concentrations. But interestingly, it is not always accumulation of ROS, which causes cell death as it could be induced by ROS and executed by specific proteins. And, it has been suggested that ROS signaling is usual in the cells, but the accumulated damage in them is quite rare (Noctor and Foyer, 2016). Another suggested possibility is tissue-specific generation of the ROS where the ROS scavenging agents are more or less inhibited during early signaling

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(Shabala et al., 2015). Annexins are also believed to have redox regulatory capacity through which they would be able to alleviate oxidative stress (Gidrol et al., 1996; Jami et al., 2008;

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Konopka-Postupolska et al., 2009) with their possible involvement in ROS-mediated signaling under stress (Laohavisit et al., 2013; Qiao et al., 2015). The ROS scavenging capacity

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plant annexins is at a lower level and it does not belong to the level that is associated with main stream antioxidants, which mainly regulate the ROS level in the subcellular ROS hubs

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like mitochondria and chloroplast. The cytosolic prevalence of annexins and their association

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with plasma membrane and other endomembranes suggest the possibility that annexins with

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the help of other proteins might be able to regulate ROS levels and generate various signatures

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of ROS, which could possibly activate various downstream signaling cascades. The subcellular distribution pattern of annexins is also an important factor in deciding the differential responses under stress conditions. Different studies on plant annexins revealed that

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annexins showed a reversible (membrane ↔ cytoplasm) distribution pattern under different stress conditions (Lee et al., 2004; Qiao et al., 2015; Wang et al., 2015). It was also suggested

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that phosphorylation could be the possible mechanism for this subcellular distribution (Deora et al., 2004; Rescher et al., 2008). Besides their subcellular distribution, annexins also localize

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to the apoplastic region and other extracellular regions like phloem sap (Giavalisco et al., 2006). The secretory nature of annexins is further supported by their calcium and plasma membrane binding, F-actin binding, GTP-binding properties, which are necessary for regulating and directing their secretion in plant cells (Konopka-Postupolska et al., 2011). The

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presence of annexin in phloem sap could be a hint towards its possible involvement in distant signaling as well. Interestingly, Thieme et al. (2015) reported in a recent study the presence of AnnAt1 and AnnAt3 transcripts in distant tissues in relation to their expression sites, and this study also proposed a bidirectional transport of AnnAt1 mRNA from root to shoot and shoot to root; however, AnnAt3 showed only root to shoot transport. On the other hand, no protein has been detected for AnnAt1 and AnnAt3 (Thieme et al., 2015). Their findings also 6

reported the differential transport pattern of AnnAt1 and AnnAt3 mRNA under phosphorus (– P) and nitrogen (–N) starvation and full nutrition (FN) conditions (Thieme et al., 2015). Moreover, the mobile transcripts of AnnAt1 (present in root, rosette, lower stem and flower) and AnnAt3 mRNA (rosette) varied in their distribution also (Thieme et al., 2015). Conceivably, the mobile AnnAt1 and AnnAt3 mRNAs might function in signaling processes linked to coordination of growth, cell differentiation and stress adaptation of distant plant

associated with the production of functional proteins in the targeted tissues. Annexins in salt and drought stress responses

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parts. Also, these mobile transcripts can act as regulatory RNA molecules or may be

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Gene expression analysis in many plant species noticed the upregulation of annexins during salt and drought stress (Table 4). It has been observed that drought and salt stress responses overlap each other at the gene expression level and expression analyses on annexins in

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different plant species also showed similar expression patterns under salt and drought stress

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conditions (Table 4). The role of annexins in salt and drought stress responses was observed in an expression study of Medicago sativa annexin AnnMs2 under different stress conditions

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(Kovács et al., 1998). Later, Kreps et al. (2002) also reported annexin upregulation under

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different stress treatments (salt, cold and osmotic) in an Arabidopsis transcriptome analysis. Subsequently, Lee et al. (2004) reported the role of annexin in salt and dehydration stresses

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and demonstrated that independent mutations in AnnAt1 and AnnAt4 led to a salt sensitive phenotype in Arabidopsis. In another study, profiling of annexin transcript under different stress treatments showed a significant increase in the AnnAt1, AnnAt6 and AnnAt8 transcripts

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under salt and dehydration stresses in Arabidopsis (Cantero et al., 2006). Further, a detailed study on AnnAt1 suggested a major role for this annexin in the drought stress tolerance

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(Konopka-Postupolska et al., 2009). Subsequently Huh et al. (2010) also reported that AnnAt1 and AnnAt4 interact with each other and regulate salt and drought stress responses in a light dependent manner in Arabidopsis. They observed that light duration played a crucial role in

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annexin-mediated stress responses contrary to positive regulation of AnnAt1 associated drought stress tolerance under short day condition (Konopka-Postupolska et al., 2009). In line with this, AnnAt1 and AnnAt4 showed negative regulation towards drought and salt stresses under long day condition (Huh et al., 2010). Following this, a recent study about the functional characterization of AnnAt8 also confirmed its role in salt and dehydration stress tolerance in Arabidopsis (Yadav et al., 2016). Expression and characterization studies from other plant species also reported the importance of annexins in drought and salt stress responses in plants 7

(Table 4 and 5). In a related study, Loukehaich et al. (2012) demonstrated SpUSP function in association with AnnSp2 in drought stress signaling. Following this, AnnSp2 overexpression in tomato plants had enhanced their tolerance to drought and salt stresses (Ijaz et al., 2017). Interestingly, annexins from other domains of life were also checked for their role in plant stress responses. Recently a fungal annexin Epann overexpression conferred a higher germination rate under oxidative stress and stronger drought tolerance on transgenic

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Arabidopsis (Zhang et al., 2017). In an interesting study, Laohavisit et al. (2013) demonstrated that AnnAt1 is essential for root growth adaptation under salt stress condition. This adaptation signal is mediated by the

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annexin in a ROS-dependent manner by inducing Ca2+ flux inside the root epidermal cells. Ca2+, which is an important secondary messenger induces the downstream signaling events. Accumulation of SOS1 in root cells is also one of the consequences of [Ca2+]Cyt increase under salt stress (Fig. 1). Mutation in AnnAt1 affects the ROS-dependent [Ca2+ ]Cyt increase under

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salt stress resulting in decreased message levels of AtSOS1 in ΔannAt1 root (Laohavisit et al.,

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2013). The SOS1 is a Na+/H+ antiporter that plays a crucial role in maintaining ion

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homeostasis during salt stress. It has also been shown for its role in root growth adaptation

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under salt stress (Huh et al., 2002). In such scenario, it is possible that AnnAt1 is indirectly involved in maintaining ion homeostasis through the regulation of SOS1 expression and root growth adaptation under salt stress (Fig. 1). The other possible regulation of ion homeostasis

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by AnnAt1 includes its inhibitory effect on the Na+ influx and K+ efflux, which needs further investigation (Laohavisit et al., 2013) (Fig. 1). Different mechanisms involved in salt stress

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responses need detailed information on association between various signaling partners. A thorough analysis of different components upstream and downstream of annexin-mediated

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signaling under salt stress would help understand the place and role of annexins in the pathway, which could pave the way for effective genetic manipulation. Recently, Jia et al. (2015) identified AtPP2B11 as an upstream regulator of annexin mediated salt stress responses. AtPP2B11 is a member of F-Box protein family and is an SCF E3 ligase. The F-

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box proteins are key regulatory proteins in many cellular processes including hormone response, photomorphogenesis, floral development, senescence, and various signal transduction pathways (Moon et al., 2004; Sadanandom et al., 2012). However, information on F-box proteins regulating abiotic stress is limited (Jia et al., 2015). Interestingly, the AtPP2B11 works in a contrasting manner in salt and dehydration stress responses (Jia et al., 2015; Li et al., 2014). Under salt stress condition, the positive regulation of AnnAt1 expression 8

by AtPP2B11 provides a hint about the degradation of some transcriptional repressor leading to transcriptional activation of AnnAt1 directly or indirectly (Fig.2). As reported by Jia et al. (2015), the positive regulation of ABF genes by AtPP2B11 may also be responsible for the activation of AnnAt1 expression due to the presence of ABRE element in AnnAt1 promoter (Fig.2). Besides AnnAt1, the expression of AnnAt2 and AnnAt3 has also been shown to be controlled by AtPP2B11 in salt stress (Jia et al., 2015). However, Lee et al. (2004) showed that AnnAt2 does not exhibit any role in salt tolerance as the annAt2 knockouts of Arabidopsis

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showed higher germination on salt medium as compared to the wild type, while Ahmed et al. (2018) observed that the overexpression of AnnBj2 (an ortholog of AnnAt2) resulted in

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insensitivity to ABA and glucose and tolerance to salt in the native system, mustard. It would be interesting to study the functional characterization of AnnAt2 in Arabidopsis through overexpression and see the salt responsive phenotype that emerges. The AnnAt1 promoter carries DRE and ABA-responsive elements, which are responsible for the ABA-independent

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and dependent transcriptional activation of AnnAt1 respectively under salt and dehydration

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stress (Konopka-Postupolska et al., 2009). Lu et al. (2015) have identified another important

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factor (AtVKOR), which is essential for ABA-mediated annexin responses under osmotic stress. Their study also confirms that AnnAt1 expression is ABA-dependent under salt and

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dehydration stress conditions. The above two recent studies demonstrate that AnnAt1 expression can be ABA-dependent as well as independent during salt and drought stress

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responses. Moreover, there is also the possibility of a cross talk between ABA-dependent and independent annexin expression (Fig. 2).

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Interestingly, the annexin regulation of other genes can also be ABA dependent and independent. Recently Ahmed et al., (2017) reported that overexpression of AnnBj2 in the

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native system, mustard was associated with the upregulation of ABA-dependent RAB18 and ABA-independent DREB2B stress marker genes suggesting that the stress tolerance exhibited by AnnBj2 overexpression in mustard is probably controlled by both ABA-dependent and -independent mechanisms (Ahmed et al., 2017). They also found that AnnBj2 transgenic

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plants exhibited insensitivity to ABA, and the altered ABA insensitivity of AnnBj2 lines was linked to the downregulation of ABI4 and ABI5 transcription factors and upregulation of ABA catabolic gene CYP707A2. ABA insensitivity or less sensitivity or tolerance can lead to various responses in plants depending on the stage of the plants. In most studies on annexins, the ABA insensitive response was documented for the seed germination and seedling stage (Ahmed et al., 2017, 2018; Huh et al., 2010; Lee et al., 2004; Yadav et al., 2016). For example 9

Arabidopsis AnnAt8 overexpressed transgenic plants also showed reduced sensitivity to ABA during seed germination (Yadav et al., 2016). Similarly, AnnSp2-transgenic plants were less sensitive to ABA during seed germination and seedling stages (Ijaz et al., 2017). In most cases, the less-sensitive or ABA tolerant annexin transgenic plants also exhibited salt and drought tolerant phenotypes. It is known that ABA plays a very important role during seed germination. During germination process, the ABA insensitivity due to annexin overexpression could be due to a decrease in the ABA level, which can be achieved by the

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regulation of ABA synthesis and degradation genes (Ahmed et al., 2017). A previous report demonstrates that an increase in Ca2+ level during germination possibly plays an important

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role in regulating the genes involved in ABA metabolism to overcome ABA inhibition of seed germination (Kong et al., 2015). Probably, annexin also modulates cytosolic Ca2+ level to counteract the effect of ABA in seed germination. It would be interesting to investigate the mechanism of annexin mediated responses at different stages of plant growth and development

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under various environmental conditions.

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Annexin and heat stress responses

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Similar to other plant stress responses, rapid increase in [Ca2+]cyt is essential to induce heat

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stress responses in plants. It has been shown that this increase is regulated by plasma membrane-localized cyclic nucleotide-gated channels (CNGCs) (Finka et al., 2012; Gao et al.,

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2012) and phospholipase C9 (PLC9) (Zheng et al., 2012), Moreover, recent reports on heat stress responses in plants proposed annexin as an important regulatory factor of heat stress response. Interestingly, proteome and transcriptome analyses of different parts of the plants

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under heat stress condition showed upregulation of annexins (Potez et al., 2011; Qiao et al., 2015; Zhang et al., 2013). First to be identified in this series was an annexin protein from the

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embryonic axes of sacred lotus (Nelumbo nucifera) that was up-regulated by heat treatment. The ectopic expression of this annexin in Arabidopsis conferred resistance to heat stress during seed germination (Potez et al., 2011). Subsequently, Zhang et al. (2013) reported that

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the expression of an annexin protein from radish leaves showed an increase at 12 h, which was followed by a decrease at 24 h in response to 40° C heat treatment. Furthermore, Wang et al. (2015) observed the upregulation of Arabidopsis annexin-1 in microsomal proteome analysis within five min of heat treatment. Similarly, Qiao et al. (2015) also observed an upregulation of rice annexin-1 under heat stress. Interestingly, overexpression of this annexin in rice conferred heat stress tolerance at the seed germination, seedling and adult plant stages. Additionally, the information from Arabidopsis and rice annexin studies predicted the role of 10

plant annexin in Ca2+ mediated heat stress responses (Qiao et al., 2015; Wang et al., 2015). Certain evidences like the change in subcellular distribution of rice annexin-1 under heat stress and its regulation of OsCDPK 24 expression under stress condition give support to the Ca2+ dependent regulation of heat stress responses of annexins (Qiao et al., 2015). OsAnn1 relocation from cytosol to the plasma membrane under heat stress condition predicted the formation Ca2+ channels (Fig.3), which probably might work as sensors of heat stress (Qiao et al., 2015). Another distinct possibility predicted by Qiao et al. (2015) was that rice annexins

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may form complexes with kinases, particularly the CDPKs (because both annexins and CDPKs are Ca2+ sensors) (Fig.3). CDPKs are key signaling components in stress responses

Arabidopsis

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(Simeunovic et al. (2016). Similarly, Wang et al. (2015) also found in their study that Annexin-1 as an important regulator of the heat stress responses (Fig.3).

Expression analysis of heat stress responsive genes, HSFs and HSPs in Arabidopsis annexin mutants showed that Annexin-1 and Annexin-2 are essential for the early changes in the gene

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expression induced by heat shock (Wang et al. 2015). Recently, Liao et al. (2017) found that

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MYB30 transcription factor regulates oxidative and heat stress responses through annexin-

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mediated cytosolic calcium signaling in Arabidopsis and myb30 mutants showed upregulation of annexins as MYB30 was shown to bind the promoters of AnnAt1 and AnnAt4, repressing

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shock responses in plants.

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their expression. Above studies identify annexins as new components in the regulation of heat

Annexin in biotic stress responses

Plants have evolved complex sensing, signaling, and defense mechanisms to confront a broad

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range of pathogens (Chisholm et al., 2006). In some cases, the plant defense strategies are specific to the type of invading organism (e.g., fungus or a bacterium) and its pathogenic

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lifestyle (i.e., biotrophic, necrotrophic, or hemibiotrophic). It is believed that the other responses are common in various biotic stresses (Chisholm et al., 2006; Dodds and Rathjen, 2010). Core responses to pathogens may be the consequence of a cross-talk between hormone-

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related pathways (Pieterse et al., 2009; Spoel and Dong, 2008); ABA, ethylene (ET), jasmonic acid (JA) and salicylic acid (SA) and/or Ca2+ mediated signaling (Dodd et al., 2010; Schulz et al., 2013), ROS; (Torres et al., 2006) and the phosphorylation cascades (Asai et al., 2002) are the major ones among them. In many cases plant responses begin with gene-for-gene recognition of the pathogen. Pathogens produce certain virulence effectors that facilitate their recognition by plants, which carry corresponding Resistance, or R genes. This causes rapid activation of defense responses, which subsequently limit the pathogen growth. Generally, R 11

gene–mediated resistance co-occurs with an oxidative burst, which is also required for another component of the response, hypersensitive cell death (HR), a type of programmed cell death that is thought to limit the access of the pathogen to water and nutrients (Glazebrook, 2005). R gene–mediated resistance is also associated with activation of a SA-dependent signaling pathway that leads to the expression of certain pathogenesis-related (PR) proteins, which contribute to resistance. Some other plant defense responses are regulated by ET and/or JA

wounding, which are also under ethylene and/ or jasmonate control.

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dependent mechanisms. These responses show considerable overlap with responses to

Expression of many genes is controlled by SA and JA, which are mutually antagonistic. ET

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and JA are required for induced expression of some genes. In contrast, only one of these signals is required for the expression of other genes. There are also cases of negative interaction between ET and JA signaling (Glazebrook, 2005). Transcription factors play an

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important role in relaying these signaling cues and activating or repressing the genes involved in immune responses and metabolic processes (Amrine et al., 2015). Since most of the plant

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immune responses are controlled by transcription, transcriptome profiling approach has been

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widely used to identify different genes involved in these responses.

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Coincidently, annexins have also been identified in numerous plant defense expression studies (Guilleroux and Osbourn, 2004; Marathe et al., 2004; Vandeputte et al., 2007; Verica et al.,

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2004; Xiao et al., 2001). Several annexin expression studies also reported their differential regulation under various hormones, which are generally involved in plant defense signaling (Table 4). Annexin expression was found induced in host plants under infection from a wide

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range of pathogens like virus, bacteria, fungi and also observed during insect infestation. Sometimes, it was observed that the responses of annexins are nonspecific to the pathogen

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infection and probably, it is a part of general defense response, which usually happens because of the cross talk and an overlap in many defense signaling pathways (Vandeputte et al., 2007). Initial report of annexin involvement in biotic stress comes from a transcript profiling of

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Rhodococcus fascians- infected BY-2 tobacco cell suspension cultures, in which an induction of plant annexin gene Ntann12 was observed in tobacco. R. fascians is a biotrophic phytopathogen that causes hyperplastic outgrowths on the plant through the synthesis of cytokinin (Stes et al., 2010). At the beginning of the interaction, bacterially produced cytokinin, which is the main virulence factor of R. fascians is detected by the plant (Pertry et al., 2009), which elicits substantial changes in transcriptomic and metabolomic profile (Depuydt et al., 2009). Eventually homeostatic mechanisms are activated that are directed 12

toward the reduction of the cytokinin levels in the infected tissues (Stes et al., 2011). Vandeputte et al. (2007) also observed a significant increase in Ntann12 expression after five days following bud infection with R. fascians strain D188, which was similar to the observations made for BY-2 cells. They also found that Ntann12 expression was higher in D188-infected tissues than in those infected by D188-5, especially seven days post-infection. NtAnn12 is also induced to a lower extent by a strain (D188-5) that is unable to induce leafy gall formation. Additionally, this gene was also induced in BY-2 cells infected with

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Pseudomonas syringae. However, Agrobacterium tumefaciens or Escherichia coli infection did not induce the gene expression, which shows that Ntann12 gene response is not only

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specific to R. fascians (Vandeputte et al., 2007). Vandeputte et al. (2007) discussed the potential role NtAnn12 in biotic stress responses that might involve Ca2+-dependent signaling. In a study of pathogen responsive genes in citrus, two citrus species, sweet orange (Citrus sinensis L. Osb.) and mandarin (C. reticulata Blanco), respectively sensitive and resistant to

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Xyllela fastidiosa (a gram –ve phytopathogenic bacerium) were used to study their genetic

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responses to the presence of X. fastidiosa. Annexin was also among those differentially

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expressed genes, which became up-regulated in mandarin infected with X. fastidiosa. In another transcriptome analysis of a Candidatus liberibacter americanus infected citrus plants,

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an Arabidopsis annexin-4 homolog was identified to be differentially expressed (Mafra et al., 2013).

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Annexins reports from eukaryotic pathogen like oomycetes and fungi are limited. Their role in tolerance towards an oomycetes pathogen Phytophthora parasitica var. nicotianae (Ppn) in

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tobacco was first observed by Jami et al. (2008).

P. parasitica is a hemibiotroph, and it

initiates infection in biotrophic mode causing little damage to host tissues. Once colonization

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has been established, it switches to the necrotrophic mode of growth (Jiang and Tyler, 2012). Biotrophic phase is short in P. parasitica and exists for only few hours (Fry, 2008; Tyler, 2007). In their study Jami et al. (2008) showed that ectopic expression of Brassica juncea annexin-1, AnnBj1 in tobacco conferred tolerance to the Ppn infection. In the detached leaf

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assay, leaves of the transgenic plants showed delayed disease development with smaller necrotic lesions ten days after inoculation. Ppn is a root pathogen of tobacco and the infiltration of tobacco leaves with CBEL glycoprotein (Cellulose-Binding Elicitor Lectin) from Ppn resulted in local necrosis of the infiltrated area and also induced various defense responses (Mateos et al., 1997; Séjalon-Delmas et al., 1997). Due to the unavailability of any transcript profiling of annexin during Ppn infection in tobacco, it is difficult to predict that 13

whether the tolerance shown by AnnBj1 in tobacco towards Ppn is specific to Ppn or a generalized defense response.

Also, the plants higher levels of message for several PR

proteins (PR-1, chitinase and glucanase) prior to pathogen attack give more support to the nonspecific defense response of AnnBj1. Three signaling pathways SA, JA and ET are involved differentially in the induction of necrosis and defense by Ppn derived elicitor CBEL (Khatib et al., 2004). AnnBj1 response towards the constitutive upregulation of PR gene transcripts is also induced by CBEL through SA dependent pathway and is a generalized

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response exhibited by the AnnBj1 transgenic plants. However, due to the unavailability of transcript data of SA, JA and ET responsive genes in the AnnBj1 transgenic tobacco after the

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pathogen attack (Jami et al., 2008), it is difficult to conclude substantively about AnnBj1 specific response towards Ppn infection.

In another study, an annexin gene from Cynanchum komarovii CkANN enhanced tolerance to

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Fusarium oxysporum in transgenic cotton (Zhang et al., 2011). Although often considered a necrotroph, the means by which F. oxysporum infects and promotes disease suggests that it is

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better considered a hemibiotroph (i.e. pathogen that lives a part of its life as a biotroph, and the

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other part, often associated with the later stages of infection, as a necrotroph or saprophyte;

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Agrios, 2005) (Thatcher et al., 2009). Though Zhang et al. (2011) observed a significant reduction in disease index values for all transgenic lines at 14 days post-inoculation (dpi) and 28 dpi, it is difficult to conclude from their observations that whether the plants were tolerant

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to the Fusarium at early stages of infection, and annexin was helpful during the biotrophic phase of the hemibiotrophic infection process of F. oxysporum or at the necrotrophic phase,

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where host cell death and lesion development occurs. However, CkANN was able to provide resistance to F. oxysporum by preventing the pathogen from completing its full cycle of its

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hemibiotrophic infection process. Increase in PR genes, PR-1, chitinase and glucanase transcript levels in CkANN transgenic cotton suggests the involvement of PR mediated SAdependent defense responses, which could be involved in the early stages of infection, which is biotrophic. But, because of the unavailability of time point data for the gene expression

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after inoculation with pathogen, it is again difficult to predict phenomenon involved in the resistance process. Interestingly, an upregulation in annexin was also observed in the root proteome of chickpea during compatible and incompatible interaction between chickpea and Fusarium oxysporum f. sp. ciceri Race1 (Foc1) infection (Chatterjee et al., 2014). In this study,

the annexin

expression was observed in the early stages of infection, which hints towards its involvement 14

during the biotrophic phase of infection. It was proposed that the enhnaced accumulation of annexins in both compatible and incompatible interactions predicts the role of Foc1 in triggering pH alterations as well as ABA driven calcium oscillations during infection that needs to be investigated (Chatterjee et al., 2014). Since Ca2+ plays a major role in signal transduction in response to various stimuli, the ability of annexin to modulate Ca2+ mediated signaling in response to different stimuli could also be crucial for annexin mediated biotic stress responses. ABA responsive protein was reported to be involved in PR-protein induction

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and disease resistance in other related studies (Choi and Hwang, 2011). It is always a complex cross talk and induced hormonal changes, which regulate disease and the plant response, with

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outcome becoming dependent on pathogen lifestyle and the genetic composition of the host (Robert-Seilaniantz et al., 2011).

In an expression study by Zhao et al. (2009), two annexins (BN18917 and BN15878) showed

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upregulation in their transcript levels during Scletoria sclerotiorum infection in Brassica napus. S. sclerotiorum, is an ubiquitous necrotrophic soilborne fungus that is among the most

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nonspecific of plant pathogens (Purdy, 1979). In a recent proteo-metabolomic study of

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transgenic tomato overexpressing oxalate decarboxylase, Ghosh et al. (2016) identified tomato

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annexin-2 as highly expressed protein during Sclerotinia rolfsii infection in E8.2-OXDC tomato fruits. Interestingly, they correlated the abundance of actin and annexin as primary strength-conferring elements in E8.2-OXDC tomato fruit that might help assemble the

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polymer networks contributing to fruit potency and increased storage capacity (Ghosh et al., 2016). Expression studies by Souza et al. (2007) in sweet orange and Puthoff and Smigocki

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(2007) in Beta vulgaris reported annexin induction during insect infestation. The possible involvement of annexins in biotic stress tolerance could also be due to their capacity to

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scavenge reactive oxygen species that are shown to be involved in several studies on plantpathogen interactions. Callose has been known to play important roles in plant growth and development in response

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to multiple biotic and abiotic stresses (Chen and Kim, 2009 and references therein). When deposited in plasmodesmata (PD), it regulates cell to cell movement of molecules that is controlled by their size exclusion limits (Iglesias and Meins, 2000). Also, callose plugs the PD and maintain bud dormancy (Rinne and van der Schoot, 1998). The deposition of callose plug or papillae at the site of wounding has been known to impede the entry of the fungal pathogens (Ryals et al., 1996); however, it is not universally accepted (Nishimura et al., 2003; Stone and Clarke, 1992) as depletion of callose synthase (gsl5) marginally increased the 15

penetration of Blumeria graminis in Arabidopsis plants. Plasma membrane–bound plant callose synthases activity is often dependent on Ca2+ (Qadota et al., 1996). Annexins are well known for their Ca2+ binding domains and thus, can reduce the availability of Ca2+ ions to callose synthase for its activity. Interestingly, callose synthase activity was found to be inhibited by 34-KD annexin in cotton facilitating elongation of cotton fibers. Surprisingly, depletion of callose in gsl5 mutants enhanced plant

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resistance against pathogens showing the adverse effect of callose deposition on plant defense system against biotic stress (Jacobs et al., 2003). Jacobs et al. (2003) also suggested that callose can either facilitate nutrient uptake by haustoria or serve as a pathogen-induced

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protection barrier preventing recognition of pathogen-derived molecules such as fungal wall polysaccharides or secreted proteins by the host. In a study, the lack of callose in the pmr4 mutant enhanced SA signaling, which resulted in increased resistance to pathogen suggesting

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that pathogen-induced callose negatively regulates the SA signaling pathway in plants (Nishimura et al., 2003). Therefore, by inhibiting the activity of callose synthase, annexins can

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enhance the defense response of plants. Moreover, the expression of Arabidopsis AnnAt1 and

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AnnBj2 was also found to be upregulated in response to salicylic acid. A significant

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upregulation in the expression of Arabidopsis AnnAt4, tomato AnnLe34, and tobacco AnnNt12 has also been demonstrated during pathogen attack (Truman et al., 2007; Vandeputte et al., 2007; Xiao et al., 2001). These studies clearly demonstrate the importance of the activity of

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annexins not only in abiotic stress responses in plants, but also in biotic resistance responses. However, studies on the role of annexins in biotic stress responses have not received much

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attention as against their involvement in abiotic stress tolerance.

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Deployment of annexins in crop improvement Initial research on plant annexins has witnessed their identification from different crops like tomato (Boustead et al., 1989; Calvert et al., 1996), capsicum (Hofmann et al., 2000), cotton (Shin and Brown, 1999), alfa alfa (Kovács et al., 1998), Medicago truncatula (de Carvalho

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Niebel et al., 1998), wheat (Breton et al., 2000), maize (Battey et al., 1996) tobacco (Seals and Randall, 1997). Annexins are presumed to be one of the important stress-responsive candidate genes that were identified in search of multiple stress-related proteins in the plant body. Although several reports suggested the involvement of annexins in plant abiotic stress and development, they did not get the proper surveillance that was needed to identify and validate the stress responsive roles of these proteins in different crop plants. This is in part due to the

16

fact that the genome sequence of many crops was not annotated. The avalanche of crop genome sequencing encouraged the identification and expression profiling of this gene family in different crops like tomato (Lu et al., 2012), soybean (Feng et al., 2013), yellow mustard (Yadav et al., 2015), potato (Szalonek et al., 2015), cotton (Huang et al., 2013), peanut (He et al., 2015), turnip (Yan et al., 2015), Chiifu (Kwon et al., 2016), strawberry (Chen et al., 2016), rice (Jami et al., 2012b) and wheat (Xu et al., 2016). The stress-responsive and tolerance roles were partly validated by raising independent transgenic plants carrying one of the members of

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annexin gene family through functional genomics. The involvement of annexins in different stresses and their characterization in transgenic crop plants are listed in Table 4 and 5.

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When we look for detailed validation of annexins in plant development and stress responses in crops, cotton annexins lead the path. Until now, many cotton annexins have been extensively characterized for their roles in cotton fiber development (Andrawis et al., 1993; Arpat et al.,

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2004; Huang et al., 2013; Li et al., 2013; Liu et al., 2016; Tang et al., 2014; Wang et al., 2010; Zhang et al., 2016) and also to some extent in abiotic stress tolerance (Zhang et al., 2015a).

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Cotton yield is determined by its fiber yield and length, and studies showed that annexins are

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promising candidates for the improvement of cotton yield and fiber quality through genetic

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engineering approaches. Being a non-edible crop, the genetic manipulation of cotton has been accepted in India and various other countries and some boll worm resistance related genetically modified (GM) verities are currently being grown in India and elsewhere in the

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world. The deployment of annexins would likely augment the future requirement of the cotton industry. This goal might be achieved through gene stacking for introducing multiple

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traits related to yield and stress tolerance. Though mportant Solanaceous crops like tomato and potato are grown across the world, their

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production in the arid regions is limited by high temperature, which causes male sterility and ultimately loss of productivity. The role of annexin

in pollen development and pollen

germination (Zhu et al., 2014a; Zhu et al., 2014b) and also in heat stress tolerance (Chu et al.,

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2012; Liao et al., 2017; Qiao et al., 2015; Wang et al., 2015) could be promising in alleviating these stresses. Amongst agronomical important crop plants, legume crops are known for their nutritive value that play very important roles in human nutrition as well as serve as supplement to improve growth of livestock (Lavin et al., 2005). Besides, they also fix atmospheric nitrogen enhancing soil fertility and boosting the yield of subsequently grown crops (Ferguson et al., 2010).

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Members of legume family fulfill their nitrogen requirement from nitrogen fixing bacteria, which live in their root nodule in a symbiotic relation. The nodulation process is regulated by various signal from plants and bacteria. Nod factors are also among these, which regulate the nodulation process. Interestingly, Mtann1 and Mtann2 coding Medicago truncatula annexin 1 and 2 respectively have been shown to be associated with symbiotic interactions, in Rhizobium inoculated roots and in nodules of M. truncatula (Carvalho‐Niebel et al., 2002; de Carvalho Niebel et al., 1998; Manthey et al., 2004). Mtann1 has been proposed to be involved

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in the early stages of Nod factor signaling and in the cell cycle activation in cortical cells (de Carvalho-Niebel et al., 1998, 2002). Further, this hypothesis was supported by the structural

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and functional characterization of annexin 1 from Medicago truncatula (Kodavali et al., 2013). Mtann2 was also found to be expressed in arbuscule- containing cells of mycorrhizal roots, and a role of the protein encoded by this gene in the membrane traffic was hypothesized (Manthey et al., 2004). Interestingly, Carrasco-Castilla et al. (2018) found in a recent study

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that the down-regulation of a Phaseolus vulgaris annexin impairs rhizobial infection and

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nodulation.

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Brassica species and their different morphotypes are used worldwide as vegetables and oilseed

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crops. The model Arabidopsis plant also belongs to Brassicaceae and is a close relative of the crop Brassicas. The study of Arabidopsis annexins opened anavenue for the annexin study in

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different Brassica members, which was further hastened by the recent genome sequencing of Brassica rapa. The initial study on Brassica annexin was done on annexin-1 AnnBj1. AnnBj1 was the first annexin to be functionally validated in both abiotic and biotic stress tolerance

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(Jami et al., 2008; Jami et al., 2009) and its ectopic expression in cotton for yield related attributes under the conditions of stress also gave very promising results (Divya et al., 2010).

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Later AnnBj3, has been characterized for its role in the methyl viologen mediated stress tolerance (Dalal et al., 2014a). Recently AnnBj2 has been characterized for its role in salt tolerance in mustard and tobacco transgenic plants (Ahmed et al., 2017,2018). Further characterization of the remaining B. juncea annexins is needed for a thorough understanding

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of the functional redundancy and diversity of this family in B. juncea. The identification of annexins in Arabidopsis and their expression analysis have facilitated the identification of annexin gene family in other members of Brassicaceae family. In a recent attempt at genomewide identification and expression analysis of Brassica rapa annexins, Yadav et al. (2015) identified an annexin member (Bra034404) of the B. rapa genome, which exhibited a very high fold increase in the transcript level under salt, methyl jasmonate, salicylic acid, H2O2 and 18

methyl viologen treatments. Subsequently, Kwon et al. (2016) also reported higher expression of Bra034404 at the protein level in the Brassica rapa. These reports showed that Bra034404 is responsive towards multiple stresses. However, further investigation is needed to understand its stress responsive mechanism and function in B. rapa. In Comparison with dicotyledonous crops, there are fewer reports on the identification and characterization of annexin gene family in monocot crop species. The first report of annexin

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identification from a monocot plant came from the isolation and expression studies of annexin (p33 and p35) in maize (Battey et al., 1996; Blackbourn et al., 1991). However, the first initiative of the genome-wide identification of annexins in any monocot crop plant was taken

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up by Jami et al. (2012b) for the identification of annexin family in Rice. Further, it was followed by other monocot crops including maize (Zhang et al., 2015b; Zhou et al., 2013) and wheat (Xu et al., 2016). The recent transcriptional analysis of annexin family in wheat gave a

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clue to their diverse roles in plant and also expedites a new approach towards annexin research related to cold-induced male sterility in wheat (Xu et al., 2016). Interestingly, the research on

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annexins in model monocot crop plant- rice is attaining pace after its genome wide

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identification in rice by Jami et al. (2012b). This study led to the identification of heat

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responsive annexin, Os02g51750, which showed upregulation under heat stress. In addition, Os08g32970 not only showed upregulation in heat stress, but also became upregulated under salt and dehydration stresses. In a recent study, Qiao et al. (2015) functionally characterized

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Os02g51750 (OsANN1) and confirmed its role in heat stress tolerance in rice. The functional characterization of different annexins in crop plants showed their involvement in stress

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tolerance (Table 5). Also a patent (US 8878006 B2) for manipulation of yield related traits in plants through the expression of an annexin is available and this invention provides a method

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for increasing yield, especially seed yield of plants relative to control plants by modulating gene expression, preferably increasing annexin expression in a plant (Lee et al., 2014). Interestingly, these potential stress tolerance functions of annexins can be advantageous for plants with respect to their deployment in crop improvement programs. Altogether, these

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findings project annexins as potential candidates for the crop genetic manipulation for different purposes including enhancement of stress tolerance, enhancement of crop yield and improvement of quality. Future prospects

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The physical basis of Ca2+ and membrane binding property for annexin has been well established. However, there are many other functions, which have been proposed for annexins (Table 2 and 3) and their physical attributes are yet to be characterized fully; peroxidase activity of annexin proteins is one of them. Despite many previous attempts to identify the physical basis, conclusive data is not available for this activity. The second one is the proposed channel property of annexins. Recent studies have emphasized on the channel property of annexins (Table 3). There is sufficient evidence, which supports their ion conductance

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property. However, the physical basis for this behavior needs to be resolved for its universal acceptance as a channel protein. Likewise, the structural or physical basis for the interaction of

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annexins with newly identified signaling components is largely unknown (Table 2). Furthermore, the identification of different interacting partners and the sites responsible for their interaction should also be analyzed in detail. As discussed earlier, the possibility of the presence of different isoforms of a single annexin, which can be targeted to different sites

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performing different functions at the spatio-temporal level needs to be confirmed. The

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identification of these isoforms may facilitate a better understanding of its multifunctional

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nature. Different proteomic approaches (phosphoproteomic and others) are now being used to identify protein isoforms (Liu et al., 2016). Moreover, it may also help in the identification of

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different post-translation modifications, which might be responsible for the targeting of annexins to various subcellular locations as well as for their functional diversity.

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It is well documented that annexins are multifunctional proteins. Last two decades of research activity has witnessed several studies on plant annexins towards their functional

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characterization (Table 5). However, many of them were focused on their role in abiotic stress tolerance in plants. Contrary to this, there are only scanty reports on the involvement of

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annexins in biotic stress responses (Jami et al., 2008; Zhang et al., 2011). Our knowledge of annexin signaling in abiotic stress is still very limited. Similarly, the mechanism behind their participation in biotic stress tolerance has not been clearly understood. Constant enrichment of our knowledge on signaling processes might help assign roles for different plant annexins. At

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the same time, it is difficult to generalize their role as of now. Most of the studies claimed the role of annexins as positive regulators of stress responses. Nonetheless, there are few studies, which also show their role as negative regulators of stress responses (Huh et al., 2010) With the availability of genome sequences of different species, the identification of the annexin families among the plant kingdom is being achieved more rapidly. Recently, Clark et al. (2012) tried to classify the annexin members in more than forty different subfamilies based 20

on their sequence analysis. In the last few years, there have been many new genome sequences published that resulted in continuous expansion of the plant annexin family. Therefore, there is a need for regular inclusion of new members in the existing classification. Moreover, some of them show new features, which have not been reported earlier for the typical annexins (Fernandez et al., 2017; Yadav et al., 2015). Such annexins need to be analyzed in detail. Conflict of interest

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The authors declare no conflict of interests with regard to the manuscript. All the authors read the manuscript.

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Author contributions

DY and PBK are responsible for the study concept, design, analysis and drafting of this article. DY, PB, and IA, contributed in literature collection and writing of the manuscript.

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Acknowledgements

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The work was carried out under a research grant from the University Grants Commission, Government of India in the form of a Project (F. No. 42-224/2013) to PBK. The authors are

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grateful to the Head, Department of Plant Sciences for facilities. DY and IA acknowledge the

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research fellowships from the CSIR and DBT, Government of India.

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Table 1: Spatio-temporal regulation of annexin gene expression in various plant species

Gene

Expression pattern

Solanum lycopersicum

AnnSl6

Specifically expressed in the stigma or (Lu et al., 2012) ovary and the ethylene treatment strongly induced expression level Specific theexpression observed in the stamen Transcript levels upregulated during the fruit ripening Expressed in mature pollen (Noir et al., 2005)

Soybean

annexin

Solanum tuberosum

Annexin1d

AnnAt1 MtAnn2

Expressed in mature pollen & germinated pollen Expressed in arbuscule-containing cells of mycorrhizal roots expressed in the fresh and raisined grapes fruits Differentially expressed in cells of soybean suspension culture Higher protein level in shoot tip

(Zou et al., 2009) (Manthey et 2004) (Briz-Cid et 2016) (Miernyk et 2016) (Bündig et 2016)

al., al., al., al.,

A

CC E

PT

ED

M

Annexin D1

SC R

AnnAt1

U

Arabidopsis thaliana Arabidopsis thaliana Medicago trancatula Tintorera red grapes

N

AnnSl4

A

AnnSl8

Reference

IP T

Plant

35

Table 2: Identified interacting partners of annexins in plants Plant

Gene

Interacting Partner

References

Annexin

Interacts with HbTCTP (translationally controlled tumor protein)

(Deng et al., 2016)

Rubber tree (Hevea brasiliensis)

OsAnn1OsAn Interacts with OsCDPK24 n1

(Qiao et al., 2015)

Gossypium hirsutum L. acc W0

GhFAnnxA

GhFAnnxA interacts with actin and these are co-localized in the cytoplasmic-nuclear Region

Gossypium barbadense

GbAnn6

AnxGb6 regulates fiber elongation through its interaction with actin

Oryza sativa

receptor-like kinase (RLK) (Os01g02580), Annexin Sterile 20 (Ste20)-like kinase (Rohila et al., (Os05g31750 (Os10g37480), SPK-3 kinase (Os01g64970) 2006) ) and casein kinase

Arabidopsis thaliana

AnnAt4

SYP121, SYP122, SYP123, SYP21 and SYP22

Solanum pennellii

AnnSp2

SpUSP

IP T

Oryza sativa

SC R

(Zhang et al., 2016)

(Fujiwara et al., 2014) (Loukehaich et al., 2012)

A

CC E

PT

ED

M

A

N

U

(Huang et al., 2013)

36

Table 3: Channel properties of annexin Gene

Channel property

Reference

Zea mays

ANN33 ANN35

Acts as Ca2+ permeable channel

(Laohavisit et al., 2009)

Capsicum annuum

ANN24

Involved in passive Ca2+ transport

(Hofmann et al., 2000)

Arabidopsis thaliana

AnnAt1

Forms K+ permeable channels in bilayers, with channel formation, favored at low pH

Arabidopsis thaliana

AnnAt1

Salinity-Induced calcium signaling and root adaptation in Arabidopsis require the calcium regulatory protein Annexin1

Arabidopsis thaliana

AnnAt1

Radical-Activated Plasma Membrane Ca2+- and K+Permeable Conductance in Root Cells

(Laohavisit et al., 2012)

Arabidopsis thaliana

AnnAt1

Regulates the H2O2-induced calcium signature in Arabidopsis thaliana roots

(Richards et al., 2014)

AnnMt1

Facilitates, the transport of Na+, K+, Ca2+ ions across (Kodavali et al., the lipid bilayer and may play an important role in 2013) Nod factor signaling

(Gorecka et al., 2007)

N

U

SC R

(Laohavisit et al., 2013)

A

CC E

PT

ED

M

A

Madicago truncatula

IP T

Plant

37

Table 4: Annexin responses to various environmental cues Gene

Response to various treatments

References

Brassica rapa

Annexins

Differentially expressed under hormonal and abiotic stress treatments

(Yadav et al., 2015)

Brassica rapa

Bra008737, Bra034402, Bra036764

Differentially expressed in shoots under drought stress

(Kim et al.)

AnnBj3 AnnBj2

Significant upregulation observed with ethephon, MJ, and wounding

AnnBj7

Upregulated on ABA and NaCl treatments

AnnAh3

Significant Upregulation by cold treatment

U

Arachis hypogea

Upregulated on NaCl treatment

BrAnnexin4

Involved in UV-A induced anthocyanin synthesis

Brassica rapa sub sp Tsuda

BrAnnexin1 BrAnnexin2 BrAnnexin3 BrAnnexin4

Significantly up-regulated under the red or far-red light treatment and differentially expressed under various abiotic stresses

Oryza sativa

Annexins

A

M

ED

(Yan et al., 2015)

(Jami et al., 2012b)

Annexins

Differentially expressed under salt and PEG treatment

(Zhang et al., 2015b)

Annexins

Differentially expressed under various abiotic stresses

(Cantero et al., 2006)

PT

Differentially expressed under various abiotic stresses

A

CC E

Arabidopsis thaliana

(He et al., 2015)

N

AnnAh1,5,6

Zea mays

(Jami et al., 2009)

SC R

Brassica juncea

IP T

Plant

38

Arabidopsis thaliana

Annexin

Upregulated at protein level in Arabidopsis shoot on NaCl treatment

(Aghaei et al., 2008)

Solanum lycopersicum

Annexin p35

Upregulated in tomato roots on NaCl treatment

(Manaa et al., 2011)

GmANN1, 11, 12, and 14

Accumulated under drought and ABA stress

GmANN1, 11 and 12

Responded to high salinity

Soybean

annexin

Upregulated at protein and mRNA level upon short term NaCl treatment

Soybean

annexin

Upregulated in soybean hypocotyls upon (Sobhanian et al., NaCl treatment 2010)

Hordeum vulgare

Annexin

Accumulated in barley crown upon drought treatment

(Vítámvás et al., 2015)

Annexin

Highly accumulated in the plasma membrane of beetroot plants after NaCl treatment

(Lino et al., 2016)

Annexin1

Increased protein level during chilling stress

p39 and p22.5

Accumulated in response to cold

Trifolium pratense L

Triticum aestivum

ED

(Red clover)

TaAnn12-A TaAnn12-D

CC E

Triticum aestivum

PT

TaAnn10

IP T

(Deng et al., 2016)

May involves in the cold-induced male sterility Accumulated upon drought and cold stress

(Bertrand et al., 2016) (Breton et al., 2000)

(Xu et al., 2016)

TaAnn2

Accumulated upon NaCl stress

TdAnn6 TdAnn12

Differentially regulated on temporal and spatial scale under different abiotic stress conditions such as salt, osmotic, (Harbaoui et al.) ionic, oxidative, ABA, SA, cold and heat stress

A

Triticum turgidum L. subsp. durum cv.Mahmoudi

(Feng et al., 2013)

SC R

U

A

(Sweet potato)

N

Beta vulgaris

M

Soybean

(Almeida et al., 2016) Peach

annexin

Accumulated in fruit mesocarp in response to stress

(Giraldo et al., 2012; Nilo et al., 2010)

39

Table 5: Plant annexins confirmed for their role in plant development and stress tolerance. Plant

Gene

Role in plant

Reference

AnnAt1

Drought, salt, heat and osmotic stress tolerance in A. thaliana

Arabidopsis thaliana

AnnAt2

Osmotic stress tolerance in A. thaliana

(Lee et al., 2004)

Arabidopsis thaliana

AnnAt4

Osmotic stress response, Interacts with AnnAt1 and regulates drought and salt stress responses

(Huh et al., 2010; Lee et al., 2004)

Arabidopsis thaliana

AnnAt5

Pollen development, pollen germination, pollen tube growth

(Zhu et al., 2014a; Zhu et al., 2014b)

Arabidopsis thaliana

AnnAt8

Salt, dehydration and methyl viologen stress tolerance in A. thaliana and tobacco

Brassica juncea

AnnBj1

Salt, dehydration and oxidative stress tolerance in Cotton

(Divya et al., 2010)

Brassica juncea

AnnBj1

Salt, dehydration, CdCl2 and oxidative stress tolerance to tobacco

(Jami et al., 2008)

Cynanchum komarovii

CkAnn 

Brassica juncea

AnnBj3

Attenuates methyl viologenmediated oxidative stress in A. thaliana

(Dalal et al., 2014a)

AnnBj3

Compensate for the thiol-specific antioxidant (TSA1) deficiency in Saccharomyces cerevisiae

(Dalal et al., 2014b)

Brassica juncea

AnnBj2

Conferred salt tolerance in Brassica juncea

(Ahmed et al., 2017)

Brassica juncea

AnnBj2

Conferred salt tolerance in tobacco

(Ahmed et al., 2018)

Nelumbo nucifera

NnAnn1

Heat stress tolerance during seed germination

(Chu et al., 2012)

Oryza sativa

OsAnn1

Heat stress tolerance in rice

(Qiao et al., 2015)

Oryza sativa

OsAnn3

Role in cold stress tolerance in rice

(Shen et al., 2017)

PT

SC R

ED

M

A

N

U

A

CC E

Brassica juncea

IP T

Arabidopsis thaliana

(Huh et al., 2010; KonopkaPostupolska et al., 2009; Laohavisit et al., 2013; Lee et al., 2004; Qiao et al., 2015)

Drought tolerance in cotton 

(Yadav et al., 2016)

(Zhang et al., 2011)

40

TdAnn6 TdAnn12

Heterologous expression of these genes in yeast improved its tolerance to abiotic stresses

(Harbaoui et al.)

Gossypium hirsutum

GhAnn1

Salt and drought stress tolerance in cotton

(Zhang et al., 2015a)

Medicago sativa

Ann Ms2

Osmotic and drought stress responses in Medicago sativa

(Kovács et al., 1998)

Gossypium barbadense

GbAnn6

Cotton fiber elongation and increased root cell length

(Huang et al., 2013)

Aspergillus fumigatus

ANXC4

Anti-stress function

(Khalaj et al., 2011)

Solanum tuberosum

StAnn1

Promote drought tolerance and mitigate light stress in potato

(Szalonek et al., 2015)

AnnSp2

Conferred salt and drought stress tolerance in tomato

(Ijaz et al., 2017)

A

CC E

PT

ED

M

A

N

U

(wild tomato)

SC R

Solanum pennellii

IP T

Triticum turgidum L. subsp. durum cv.Mahmoudi

41

42

A ED

PT

CC E A

M

N U SC R

IP

Figure 1: Schematic representation of salt stress responses in the plant cell highlighting the potential roles of annexins and other proteins

IP T

Na+ enters into the cells through non-selective cation channel (NSCC), leading to the depolarization (DPZ) of the membrane. Membrane depolarization causes the activation of other NSCC to influx the Ca2+. The increased [Ca2+] Cyt activate the NADPH oxidase (NOX). The NOX

SC R

activation results in the e- transfer leading to an increase in the extracellular ROS level, which activates the ROS sensitive NSCC and annexin facilitating the increase of cytosolic Ca2+. This NOX and NSCC are supposed to be interdependent and they help in amplifying this Ca2+ influx by a loop based activation. While the possibility of annexins participation in this loop is unknown,

U

this annexin-mediated increase in the cytosolic Ca2+ was supposed to activate the SOS1

N

transcriptionally. Further, this increased [Ca2+]Cyt level is sensed by different Ca2+ binding

A

proteins (CaM, annexins) and also different Ca2+-dependent protein kinases (CDPK, CBL-CIPK). The Recent findings also support the possibility of annexins interaction with these Ca2+-dependent

M

protein kinases; together they can form a complex with the Ca2+. By this, they can regulate the downstream components, which include the phosphorylation of different transcription factors like

D

NAC, MYB, AP2/ERF, WRKY, bZIP and bHLH, leading to the transcriptional activation of

TE

different salt and osmotic stress-responsive genes. The transcriptional activation of annexins in the salt stress condition can be ABA-dependent or independent. Here, the ABF is shown as the

EP

transcriptional activator of the annexins under salt stress. The functional activation of ABF is ABA-dependent, but its transcriptional activation can be ABA-dependent or independent. DPZ:

A

CC

Depolarization.

43

44

A ED

PT

CC E A

M

N U SC R

IP

Figure 2: An overview of the potential regulatory mechanism of annexins expression in plants Recent reports on annexins in Arabidopsis (Jia et al., 2015; Lu et al., 2015) identified some upstream regulatory components. In this series, AtPP2B11 has been identified as an upstream

IP T

regulator of annexin expression under salt stress condition. AtPP2B11 responds to salt and drought stresses in an opposite manner. AtPP2B11 is a member of F-box family and its role in abiotic stress is now being identified. It is an E3 ligase and its regulatory function is possibly due

SC R

to its protein degrading properties that can lead to the removal of transcriptional repressors, which can finally lead to the expression of different genes. Here, the ABF is shown as the transcriptional activator of the annexins under salt stress. The functional activation of ABF is ABA-dependent,

U

but its transcriptional activation can be ABA-dependent or independent. LTO1/AtVKOR is

N

involved in ABA-dependent osmotic stress responses in Arabidopsis. lto1 mutant of Arabidopsis plant lacks the ABA-dependent expression of ABFs and annexin. The dependence of annexin on

A

ABF is common in both ABA-dependent and ABA-independent regulation of annexin expression

M

under drought and salt stress. However, the expression of ABF genes can be ABA-dependent or independent under salt stress condition. Possibly, there is a

D

cross talk which exists at the level of ABF during the ABA-dependent and independent stress

A

CC

EP

TE

responses.

45

46

A ED

PT

CC E A

M

N U SC R

IP

Figure 3: Potential role of annexins during heat stress responses in plant cells Studies in Arabidopsis and rice suggest that annexin can respond to heat stress by modulating the

IP T

intracellular Ca2+ flux and also by interacting with H2O2 and various other signaling components involved in heat stress response. The increase in the ambient temperature causes heat stress in the plant. Heat stress sensing mechanism has not been identified in the plant. Recent studies by Wang

SC R

et al. (2015) and Qiao et al. (2015) identified annexin as a critical component of heat stress responses in the plant. The initial Ca2+ influx from the sensor Ca2+ channels, Cyclic nucleotide gated channels (CNGCs) leads to the redistribution of annexin, which activate the annexins and other Ca2+ channels to increase the Ca2+ influx. The increase in ROS under heat shock plays a

U

major role in the activation of heat stress responses. The induction of annexin under heat stress

N

and its interaction with ROS and Ca2+ propose that it as an important modulator of downstream

A

responses. Increase in the annexin level under heat stress leads to the induction of Heat stress transcription factors (HSFs), which are essential for the transcriptional activation of heat shock

M

proteins (HSPs). Some other genes like Superoxide dismutase (SODs), Catalase (CAT) and Calcium-dependent protein kinases (CDPKs) also get induced. CDPK and annexin possibly make

D

a complex in the presence of Ca2+, leading to the activation of different transcriptional factors

A

CC

EP

TE

(unknown).

47