Response of Asplenium nidus to Drought Stress and Identification of Drought Tolerance Genes

To explore how Asplenium nidus responds to drought stress, and to mine drought-responsive genes for Asplenium nidus, we conducted a pot experiment in the IOT smart greenhouse. We measured a series of physiological and biochemical indexes for drought-treated plants, and analyzed the expression of drought-responsive genes in Asplenium nidus by RT-qPCR. The results show that the Asplenium nidus is a species with drought tolerance. Asplenium nidus inhibits plant photosynthesis and reduces life activities by limiting stomatal opening to adapt to drought. To resist drought stress, the Asplenium nidus changes the osmotic potential by increasing the proline content to maintain normal metabolic activities and prevents the damage of active oxygen by increasing the enzyme activities of SOD and POD. Based on the analysis of the relative expression levels of genes, AVP1-2 and AVP1-4 may be drought-resistant genes in Asplenium nidus. This study lays the foundation for in-depth research on drought tolerance mechanisms and drought-resistant gene mining of Asplenium nidus.


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Determination of physiological parameters: In this experiment, we measured five 119 physiological parameters: relative leaf water content(RLWC), the maximum net 120 photosynthetic rate(P max ), transpiration rate( T r ) , stomatal conductance( g s ) , and 121 intercellular CO 2 concentration(C i ) the three groups of bird's nest fern.

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The RLWC of leaves is measured every two weeks. We randomly selected three plants 123 per treatment, and each plant took a mature and healthy leaf for measurement. The 124 relative water content is measured by measuring the fresh weight (FW) of leaves 125 immediately after removing it, and then immersing leaves in distilled water for 3-4 hours.

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When the constant weight is reached, the weight of the leaves is saturated weight (TW).

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Then put leaves into an oven at 120 ° C for 30 minutes and bake it to a constant weight at

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Measurement results of physiological parameters 196 RFWC of bird's nest fern leaves in CK group has been maintained at about 90%( Figure 2).

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After drought treatment, the leaf relative water content in T1 group fluctuated between 60% 198 and 80%, and that in T2 group decreased to about 40% (Fig. 3). RFWC could be reverted 199 to the normal range after rehydration for two weeks in T1 and T2.

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The change trends of P max ,T r and g s , with different degrees of drought were similar (Fig.4).

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After drought treatment, the three indexes in T1 and T2 groups decreased to near zero, 206 only in T1 group increased slightly in the 3rd week, and increased rapidly in both groups 207 after rehydration, and the rising range in T1 group was higher than that in T2 group.

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The change trend of C i was opposite to that of the other three indexes. After drought 209 treatment, the intercellular CO 2 concentration in T1 group and T2 group was significantly 210 higher than that in CK group, and the rising range in T2 group was larger than that in T2 211 group. After rehydration, the intercellular CO 2 concentration in both groups decreased to 212 normal value immediately.

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We performed a correlation analysis on P max , T r , g s and C i of the T1 and T2 groups, 220 respectively. The analysis results showed that there was extremely significant positive 221 correlation between P max , T r , and g s in the T1 and T2 group (Fig.4), while C i was 222 negatively correlated with P max , T r , and g s , respectively. In the T1 group, there was 223 extremely significant negative correlation between C i and P max , but C i was significantly 224 negatively correlated with T r and g s . However, in the T2 group, C i was significantly negatively 225 correlated with P max , T r and g s .

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The SOD enzyme activity in the T1 group increased slightly after drought treatment and did not decrease after rehydration，while the T2 group increased significantly during the 258 2nd week of drought treatment, then gradually decreased in the later period, and 259 decreased to the normal range after rehydration (Fig.8). The POD enzyme activity in the 260 T1 group increased slightly in the 2nd and 6th week, while the T2 group increased 261 significantly in the 4th and 6th week, and they all returned to the normal range after 262 rehydration. CK, T1, and T2 had the same trend of CAT enzyme activity but T2> T1> CK 263 as a whole. All the three treatments increased significantly twice in the 4th and 8th week

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(the 2nd week after rehydration). 276 was named H_PPase in the PfamA database. This domain accounts for more than 95% of 277 the total AVP1 protein sequence. The total length of the protein sequence of AVP1 gene 278 is 770 amino acids, and its conserved domain is from 21th amino acids to 755th amino 279 acids (Fig. 9).

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The primers of RT-qPCR were designed by using Primer5 and Oligo7 software in the 295 Table2.Using EF1a as an internal reference gene, the gene expression of AVP1-1,

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AVP1-2, AVP1-3, and AVP1-4 were analyzed. It is worth noting that compared with CK, 297 the relative expression of AVP1-2 in T1 group increased significantly in the 6th week,

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while that in T2 group increased significantly in the 2nd and 4th week, and decreased in 299 both groups after rehydration (Fig.11). Compared with CK, the relative expression of 300 AVP1-4 in T2 group increased significantly in the 4th week. The expression of AVP1-1 301 and AVP1-3 was not related to the degree of drought and the time of drought.