Effect of desiccation on seed germination
Consistent with other recalcitrant seeds [14, 15, 25], the GP of Q. variabilis seeds decreased along with the decrease of MC, and there was significantly positively correlated between MC and GP. The critical MC of recalcitrant seeds were determined when about 50% seeds became unviable, which can be used as a standard of seed desiccation sensitivity [7]. In the present study, the critical MC of Q. variabilis seed was about 28.20%. Furthermore, when the MC of seeds decreased to 17.17%, the seeds lost their vitality completely, which was similar to the recalcitrant seeds of Aesculus chinensi [26].
Phytohormone synthesis
In plant, hormones play important roles in coping with abiotic stress [27]. For instance, ABA can regulate the drought stress response and is also a vital hormone for seed desiccation tolerance acquisition [28]. ABA synthesis and catabolism in plants are mainly regulated by NCED and CYP707A [29]. Similar to previous study on recalcitrant Camellia sinensis seeds [15], genes related to ABA synthesis, such as NCED, crtZ, and AO, were down-regulated, while ZEP was up-regulated during Q. variabilis seeds desiccation. Furthermore, previous research also has pointed out that lack of ABA in the ZEP-deficient mutant of Nicotiana plumbaginifolia resulted in rapid water loss [30]. In this study, although the expression of ZEP was up-regulated, the down-regulation of NCED, crtZ and AO might lead to the decrease of ABA content during seed desiccation. ABA 8'-hydroxylases encoded by CYP707A can deactivate ABA and play an irreplaceable role in ABA catabolism [31]. ABA can also be reversibly inactivated by glucosylation. For instance, plants lacking UDP glucosyltransferase have fewer glucosyl esters and more free ABA, exhibiting stronger resistance to water deficit [32]. In our study, genes encoding ABA β-glucosyltransferase were up-regulated, and genes encoding UDP glycosyltransferase were both up- and down-regulated. Therefore, we speculate that the downregulation of genes encoding NCED, crtZ, AO, ABA β-glucosyltransferase, and upregulation of the UDP glycosyltransferase related genes might cause the decline of ABA content during seed desiccation.
It is widely known that indole pyruvate pathway is the most basic pathway for IAA synthesis in plants [33]. FMO encoded by YUC family genes has been confirmed to be able to directly convert indole pyruvate to IAA [34]. In this study, we found that YUC family gene (LOC112010754) was up-regulated, which may lead to the increase of IAA content during Q. variabilis seeds desiccation.
ABA and IAA are closely related to many physiological processes in plants and their interactions regulating to various developmental processes such as seed dormancy [35], lateral root development [36], and plant growth [37]. For instance, during the seed maturation of Coffea canephora, the up-regulated expression of many genes related to IAA may inhibit the mitochondrial retrograde response while ABA has the opposite effect, playing an important role in the acquisition of desiccation tolerance [38]. In this study, desiccation leads to the differential expression of genes related to hormone synthesis and catabolism, and changes the content of ABA and IAA. Thus, the balance between hormone is changed, which has been demonstrated to be able to induce desiccation tolerance in desiccation sensitive seeds, i.e., Acer saccharinum and C. limon seeds [13, 39]. Therefore, the desiccation sensitivity in recalcitrant Q. variabilis seeds might be a result of a broken balance between ABA and IAA.
Plant hormone signal transduction
Plant can respond to stress by regulating the expression of genes in the ABA signalling pathway, which is critical for plant response to water deficit [40]. ABA receptors PYR/PYLs, PP2Cs, SnRK2s, and ABF constitute the core network of ABA signal regulation [27, 40]. In plants, the perception of ABA through PYR/PYL receptors is necessary for ABA signal transduction. The PYR can interact with ABA, inactivate PP2C, activate SnRK2 and self-phosphorylation, activate ABF and then regulate the expression of related stress genes [41, 42]. During desiccation, the downregulation of PYL, PP2C, and SnRK may cause recalcitrant tea seeds sensitive to desiccation [15]. In our study, desiccation induced downregulation of PYR, PP2C and ABF, while SnRK2 were both up- and down-regulated. These results indicate that desiccation interferes with the ABA signal transduction pathway, which eventually results in high desiccation sensitivity.
The TIR1/AFB-AUX/IAA/TPL-ARFs pathway is a widely accepted IAA signalling pathway. IAA can promote the formation of synergistic receptor complexes between TIR1 and AUX/IAA proteins, thereby activating TFs of auxin response factor (ARF) family and regulating IAA response genes [43]. In the present study, the expression of AUX/IAA genes were both up- and down-regulated, that of the ARF genes were down-regulated, and that of SAUR gene was up-regulated, similar to the findings in C. canephora seeds [38].
The auxin response factor2 (ARF2) in Arabidopsis negatively regulates the homologous domain gene HB33, which is a regulator of ABA signalling pathway [37]. As previously reported, IAA can activate its response factor ARF10/16 to induce the expression of ABI3, thus activating the ABA signal transduction pathway [35]. Moreover, the PYL8 can enhance the expression of the IAA response genes by activating the WRKY77 transcription factor [36]. In the present study, desiccation induced differential expression of ARF and PYL in hormonal-signalling pathway in Q. variabilis seeds, indicating the potential interaction between them might contribute to regulate seed desiccation sensitivity.
It has been demonstrated that members of MYB, WRKY, ERF, NAC, bZIP, and bHLH TF families are key regulatory factors in plant response to water stress [44]. In this research, the screened DEGs contain ERF, bZIP, bHLH and other TF families. And the ERF1B, ABI5, TGA9, TGA10, UNE10, PIF3 and PIF5 from screened families were involved in "plant hormone signal transduction" pathway. Among them, ERF1B protein play an important role in tolerance to abiotic stress [45]. Furthermore, it has been reported that ABI3 is essential for acquisition of seed desiccation tolerance [16, 28]. The ABI3 can interact with ABI5 to regulate the expression of downstream genes and mediate ABA signal transduction [46]. In our research, the ERF1B was up-regulated and ABI5 was down-regulated, which may change the expression of downstream stress-related genes and result in seed desiccation sensitivity. In addition, our study also found that GATA and Trihelix TFs were differentially expressed during seeds desiccation, which were reported to play crucial roles in plant response to abiotic stress [47, 48]. These TFs need to be further explored, in order to provide reference for understanding the molecular regulatory mechanism of recalcitrant Q. variabilis seeds desiccation sensitivity.
Protective proteins related to desiccation
HSPs play irreplaceable roles in protecting plants from abiotic stress [49] and are particularly related to the acquisition of seed desiccation tolerance [13]. There is a 22 kDa HSPs in recalcitrant Acer saccharinum seeds, and its content increases significantly after desiccation, so as to reduce the damage [50]. In this research, we identified eight kinds of HSPs with molecular weight of 15.7, 17.5, 18.1, 18.2, 18.5, 22.0 and 22.7 kDa, as well as HSP70, which were mainly up-regulated during Q. variabilis seeds desiccation. This result is consistent with recalcitrant T. chinensis seeds [14]. And we also identified that heat shock transcription factors (Hsfs) were differentially expressed during desiccation, which play crucial roles in plant response to abiotic stress [51]. In addition, LEAs and the transcripts encoding LEAs were reported to accumulate in large amounts during acquisition desiccation tolerance of orthodox seeds [52], which can prevent protein aggregation during desiccation by folding [53]. While recalcitrant seeds have fewer LEAs or the absence of specific LEAs [54]. In Castanospermum australe and Medicago truncatula seeds that are sensitive to desiccation, the seeds are unable to accumulate enough LEAs, which may contribute to their sensitive to desiccation [46]. In this study, LEA-related genes were mainly down-regulated during seed desiccation, which may inhibit the capability of Q. variabilis seeds to accumulate LEAs to resist the damage. In addition, ABI3 indirectly induced the desiccation tolerance of seeds by regulating the expression of HSPA9 to the increase of HSPs content, both ABI5 and ABI3 also connect many LEAs as well as desiccation tolerance related genes in M. truncatula seeds [55]. In the present study, ABI5 may regulate the expression of HSPs and LEAs related genes to cope with the adverse effects of desiccation. However, the specific regulatory relationship between them needs further experimental analysis.
Glycerophospholipid metabolism
Membrane phospholipids proportions are different between recalcitrant and orthodox seeds [56, 57]. Phospholipase D (PLD) can hydrolyse phospholipids into phospholipid acid (PA) and is closely related to desiccation sensitivity, i.e., the increased of PA content and decreased seed survival under desiccation [58]. Furthermore, PA also can be induced by ABA, and as the second messenger to participate in plant responses to abiotic stress [40]. In this study, several ABA-related genes were differentially expressed, and we also found that PLD related genes were up-regulated. Therefore, PLD may become more abundant and increase PA content during desiccation, resulting in reduction of the membranes fluidity, leading to the desiccation sensitivity of Q. variabilis seeds.