1. IntroductionGrapevine (Vitis vinifera L.) is a perennial fruit crop that is cultivated globally with 8110 varieties [United States Department of Agriculture Foreign Agricultural Service 2022, United Nations FAO, 2019] [1]. In 2020, viticulture occupies an area of 7.3 million ha, with around 85 million tonnes of table grapes, 1.4 million tonnes of dried grapes, and 260 million hL of wine being produced worldwide [International Organization of Vine and Wine, OIV, 2019]. However, in the prosperous grapevine industry, there are increasing challenges from viruses, bacteria, fungi, pests and environmental stresses; for reviews see [2,3,4,5]. Under the frame of agroecology, the strategies for viticulture stress management have been shifted from traditional chemical control to antagonistic anti-microorganism screening and key resistant loci mining for genomics-based breeding [6,7]. The boundary between biotic and abiotic stress-induced plant defence responses are sometimes obscure; for instance, the jasmonic acide (JA) dependent default pathway is triggered by salt stress in grapevine. Their modulation by a parallel pathway activated by pathogen-derived factors has been approved [8]. These biotic/abiotic signals overlap in their upstream events (calcium influx, apoplastic alkalinisation and induction of JA signalling), but differ in their downstream output (stilbene metabolism, different phytoalexin synthesis genes and oxidative burst) [8]. Another example comes from V. rupestris affected by O-methylmellein, a conserved pathogenic factor. In this case, O-methylmellein as an amplifier of flg22 shares a specific signature depending on RboH, independent of calcium influx. This means it works neither as a candidate toxin, nor an elicitor activating basal immunity, but as a weapon in interspecies competition by amplifying the basal immune response [9].Moreover, it is helpful to refer to the classic CODIT (Compartmentalization Of Decay In Trees) and recent findings on the cellular level responses to have a better understanding of host responses to biotic/abiotic stresses [10]. Where the vascular wilt systemically spreads microorganisms or structures such as spores or hyphae, and phytotoxins are mainly restricted to the vessel lumen and cells surrounding vessels, canker is hardly able to spread systemically due to the death of a portion of the vascular cambium that disables the newly formed functional xylem and phloem. Therefore, from the plant side, the resource allocation could be subjected to a trade-off between vessel occlusion and defence reaction in conjunctive living tissues, as well as the signalling transduction cascade within single cells and amongst the symplast through plasmodesmata. The worldwide spread of Grapevine Trunk Disease has been shown to be damaging depending on fluctuating environments as the fungi can be latent in the tissues for years [11]. This may hold true for the other biotic stresses. Therefore, it is interesting to find out about some signalling to diverge the abiotic stresses from the biotic stresses, or branching points upstream or downstream of certain genes.Among these, stress-associated proteins (SAPs) have attracted much attention. SAPs are a class of zinc-finger proteins containing A20/AN1 domains, which have emerged as important components in plant stress tolerance in the past decade [12,13]. The first identified zinc finger protein was the cytokine-induced protein A20, functioning in the human umbilical vein endothelial cells [14]. This protein is induced by tumour necrosis factor (TNF) and inhibits TNF-induced apoptosis [15]. The other zinc finger domain, AN1, has been characterised for the first time in a ubiquitin-like fusion protein localised in the hemisphere of Xenopus laevis eggs and early embryos [16]. As for the C-terminal AN1 domain, it was first found in the protein encoding the hemispheric parent RNA of Xenopus laevis. The conserved sequence is CX2CX9-12CX1-2CX4CX2HX5HXC [17]. The AN1 zinc-finger domain is usually found associated with the A20 zinc-finger and such types of proteins are present in many eukaryotes [18].Though A20/AN1 domains were discovered in animals, their roles have been gradually illuminated in plants. Early studies on SAPs have been focused on abiotic stresses at the transcriptional level and gene function. The first plant A20/AN1 protein was cloned from rice (OsiSAP1) and showed enhanced expression in response to cold, drought, salt, submersion, heavy metals and injury [19]. In addition, OsSAP9 (ZFP177) and AtSAP5 were found to be induced by heat, cold and H2O2, and to play an important role in enhancing temperature stress tolerance [20,21]. Evidence from the OsiSAP family as to salinity, osmotic pressure, and hormone and oxidative stresses were reported for a review; see [22,23]. To date, a total of 18 SAPs have been isolated from rice, 14 in Arabidopsis, and 37 in cotton. SAPs have also been identified in other horticulture plant species, such as grapevine, tomato and apple [24,25,26]. SAPs interact with many other proteins to execute their functions [27]. There is direct evidence in the cross-talk between biotic and abiotic stress signalling pathways from the first identified SAP in a plant that OsSAP1 plays a key role in basal resistance against pathogen infection [28]. With respect to grapevine, the SAP gene family has been studied under abiotic stress. The overexpression of VaSAP15 rendered the transgenic plants lower in electrolyte leakage and malondialdehyde levels and higher in antioxidant enzyme activity under cold stress [29]. However, the knowledge of SAP gene family reactions under biotic stress remains largely unknown.

In this study, we identified all VvSAPs genes and explored their evolutionary relationships, sequence features and duplication events. The tissue-specific expression of grapevine SAPs among species under biotic/abiotic stresses were analysed and their potential cross-talking signalling pathways were further discussed. Those will provide references for optimizing the management practices and for mining candidate resistant loci for genomics-based breeding. The signalling pathway induced by SAPs encoding the A20/AN1 zinc protein domain will shed light on further study on the biotic and abiotic stresses inducing signalling cross-talking in grapevine.

4. DiscussionResistance to biotic and abiotic stresses is a central topic for sustainable agriculture, especially in grapevine, one of the world-wide cultivated crops between 30° and 50° N and 30° and 40° S with the highest economic output per acreage. Abiotic stresses strongly affect biotic interactions at various levels, for instance, plant morphogenesis from cells, tissues to organs, transcriptomic regulation, signalling pathways, as well as primary and secondary metabolisms. These factors combine to make an individual plant a more or less suitable host for its counterpart [36,37].Proteins containing A20/AN1 zinc fingers have been characterised as regulating the immune responses in animals and environmental responses in plants [38]. The grapevine genome database was referred to in this study, and a total of 15 VvSAP gene members was identified and characterised (Table 1). Genome-wide survey of SAP gene family composition was further performed in other plant species. The family members of SAPs in grapevine are more than in maize (11), Arabidopsis (14) and tomato (14); while less than in rice (18), potato (17), tobacco (16) and cotton (37). The conservative domain of the VvSAP family was analysed (Figure 4). It was found that all candidates contained the conserved AN1 zinc-finger domain and mostly also contained the A20 zinc-finger domain, which is similar to findings in Arabidopsis, cotton and tomato, but not in rice. In addition, there were five SAPs in rice and four in Arabidopsis with only the AN1 zinc-finger domain. On the other hand, in the evolutionarily primitive organisms such as Saccharomyces cerevisae and Aspergillus fumigatus, only an AN1 zinc finger domain, being devoid of A20 domain, has been found, and with similar properties in cotton. This may indicate the ancient origin of the AN1 zinc finger domain compared to A20 domain [12,24]. Chromosomal positions of VvSAP were identified using Tbtools1.098763 (Figure 2).Analysis of the evolutionary relationships of SAPs among grapevine and two other species indicated that these SAPs can be divided into three groups. The grapevine SAPs in group II were mostly with the A20-AN1-type, excepting VvSAP3 containing a single AN1 domain (Figure 3). The conserved motif distributions of VvSAP according to the evolutionary relationship were further examined. A total of 10 conserved motifs were identified, and their distributions exhibited strong evolutionary conservation (Figure 1). Gene structure can also effectively reveal evolutionary relationships among gene families. Zhou et al. (2018) reported that the intronless SAPs could reduce post-transcriptional processing, and therefore can be utilised to test immediate responses to abiotic stresses [39]. According to these findings, SAPs are highly conserved evolutionarily in plants [39]. The chromosomal positions of candidates were also obtained from V. vinifera genome annotation: Table 1. The location of SAP genes in grapevine can help to identify these genes in other species, directly or using microsynteny among them.Plant SAP genes are thought to play a vital role in regulating a variety of biotic and abiotic stresses according to previous studies [23,40,41]. The most well-characterised plant protein in this class is the rice A20/AN1 zinc-finger domain stress associated protein 1, OsISAP1. Mukhopadhyay et al. (2004) found that the over-expression of OsISAP1 exhibits tolerances to cold, water defection and salinity stress [19]. Moreover, the OsSAPs are responsive to multiple biotic stresses. The overexpression of OsSAP1 enhances the basal resistance against pathogen infection in tobacco [28]. Our results indicated that nearly all VvSAP genes were up-regulated in both vegetative and reproductive organs, whereas VvSAP1, VvSAP12 and VvSAP13 were down-regulated. The decreased extent was much stronger in vegetative organs than in reproductive organs. VvSAP4 and VvSAP8 exhibited a much higher transcript abundance in pollen than in other flower structures (Figure 5). Further, a native American grapevine (V. rupestris), a native European grapevine (V. vinifera ‘Cabernet Sauvignon’) and a hybrid species (V. aestivalis × V. vinifera ‘Norton’) were compared respectively for their biotic stress resistance (to Botryosphaeriaceae and Erysiphaceae) (Figure 6). Given that the expression data at the quantitative level were generated by different methods (microarray Figure 5, Figure 6C and Figure 7 vs. RNAseq Figure 6A,B), the comparisons in this study are restricted within the same method. There are significant different expression patterns (nothing to consider their quantitative level) among American and European grapevine varieties. VvSAP10 was highly induced in V. rupestris. The expression levels were more obvious in two less virulent fungi. D. seriata 98.1 and B. dothidea Sd-18. than in N. parvum Bt-67 (Figure 6A,B) [11]. The induction of VvSAP10 presented an earlier induction in V. vinifera ‘Cabernet Sauvignon’ than in V. aestivalis × V. vinifera ‘Norton’. VvSAP10 showed mild changes to osmotic and salinity stresses, but strong changes to the temperature stress (Figure 7). VvSAP6 was the most significantly enhanced gene against all of the tested abiotic stresses. Especially under osmotic stress, the VvSAP6 mediated salinity-induced resistance occurred later than osmotic stress (Figure 7A,B). The expression pattern of VvSAP1, VvSAP2, VvSAP3, VvSAP5 and VvSAP8 was down-regulated in the short term and then enhanced (Figure 7A,B), which is different to the SAPs in Arabidopsis, cucumber and cotton [20,42].Temporary water deficit stress could enhance the immunity of Arabidopsis by stomatal closure, thereby enhancing the resistance to Pseudomonas syringae pv. Tomato DC3000. In this case, a downstream gene transcription level has confirmed that the E3 ligase SINAT4 down-regulated the cysteine protein RD21A [43]. Our previous study on the actin filament (AF) marker line in grapevine indicates that guard cells act as pacemakers of defence, dominating the responses of the remaining epidermal cells. It has been shown that to the phytopathogenic bacteria P. syringae pv. Tomato DC3000, actin responses were confined to the guard cells. In contrast, upon contact with zoospores of Plasmopara viticola, not only the guard cells, but also epidermal pavement cells where no zoospores had attached, responded with the formation of a perinuclear actin basket [44]. The cold stress normally inhibits the JA-dependent signalling pathway but activates the salicylic acid (SA)-dependent signalling pathway; the high temperature brings a contrary consequence [36]. Although the gene expression levels induced by abiotic stresses are normally much milder than the biotic stress induction (Figure 6 and Figure 7), it probably can affect the activation of certain resistance genes in plants challenged by further attacks of pathogenic microorganisms [36]. Paolinelli-Alfonso et al. (2016) found that transcriptome analysis of L. theodromae in the presence of grapevine wood revealed an upregulation of genes involved in the SA pathway [45]. This upregulation was enhanced by high temperature. The high temperature-induced contradictory SA signalling pathway can be understand by taking account of the biotic induced signalling. An activation of L-tyrosine catabolism pathway could lead to inhibition of the SA pathway, favouring fungal development especially during heat stress [45]. On the other hand, high JA concentration is known to have an antagonistic effect on the SA pathway [46]. Botryosphaeriaceae are known to produce JA, which has been identified in the culture filtrate of some GTD agents, e.g., N. parvum and N. vitifusiforme [47]. Hence it could be hypothesised that, in addition to a toxic effect, JA inhibits the SA pathway involved in the stimulation of plant defences. Zhao et al. (2021) have reported a downregulation of VvWRKY70 [30], a putative central component of the SA pathway, after treatment of V. rupestris cells with D. seriata secreted compounds.These signalling cascades are partially sharing with abiotic induced defence responses, although it is to be noted that this is a matter of timing events. Otherwise, a consequence-centred explanation may lead to an apparent discrepancy. For instance, under cold tolerance study, a transient disassembly of microtubules followed by their recovery with progressive acclimation has been approved to be a kind of cold acclimation, which is obviously contradictory if to follow the final consequence that a stable microtubules array was established for cold challenging [48]. Moreover, the above discussed JA is necessary to convey efficient disassembly of MTs under cold stress, but that is not the exclusive signal. Another example comes from the cytoskeleton (actin filaments and microtubules) early arrangement (within a time frame of 20 min vs. long-time adaptation, e.g., hours or days) to distinguish the PTI from ETI [49]. This evidence could come back to answer the basic question, that environmental mediated biotic stress symptoms are not always coincidental with the invasion of casual agents.The above findings indicate crosstalk and convergence of mechanisms in these pathways and the existence of a general stress response, suggesting that resource allocation could be subjected to a trade-off between vessel occlusion and defence reaction in conjunctive living tissues in grapevine, as well as the signalling transduction cascade within single cells and amongst the symplasts through plasmodesmata. The principle of the CODIT lies in the compartmentalisation of the established four obstacles that restrict and decay pathogen spread [50]. The first three obstacles located in xylem per se can be interpreted as the reaction zone after pathogen invasion. Obstacle four, consisting of living cells, e.g., paratracheal parenchyma, fibres and ray, can protect the host via arming themselves with newly formed structures. The mystery of environment-related grapevine disease, such as the disruption of water flow, can probably be explained via vessel dependence systemically spread, as well as local living cell signalling transduction against these four obstacles [10]. VaSAP15 from a Chinese wild grapevine V. amurensis has been confirmed to localise in both the nucleus and cell membrane, which prompts a pending question during the penetration of fungi, that the contraction of the actin around the penetration site probably linked with the actin nucleus basket to attract the nucleus towards the penetration site [29,51]. Plant-specific class XIV kinesin KCH1 has been confirmed for its function as a tether [9,52]. Therefore, the VvSAPs are probably triggered at the early time point via an acting-dependent endocytotic of a receptor on the member.