YL fruits and flowers had less freezing rate under low temperature
YL is a bud mutation found from a 35-year-old RT loquat in Tongyu Street, Bututang Village, Luqiao District, Taizhou City, Zhejiang Province in 2003. After years of observation, significant differences in fruit shape, seed number, and edible rate were found between YL and its mother plant RT. For example, the number of seeds per fruit in YL is 1.03 less than that in RT (Figure S1).
Given that loquat is originally a subtropical fruit tree, freezing tolerance became the major factor of fruit production. Through years of investigations on the annual occurrence of freezing damage, bud mutation YL exhibited strong freezing tolerance than its parent RT, especially the fruits (Fig. 1a and 1c).
Under low-temperature conditions, RT flowers were susceptible to freezing damage, which was characterized by deformed flowers, incomplete differentiation of flower buds, and local browning. Thus, according to these abnormal phenotypes, the freezing flowers of RT and YL were counted. Finally, the freezing rates of YL flowers were only 1.70% at those years with freezing winter (2010, 2014 and 2017), whereas those of RT were 6.30% (Fig. 1d).
At the fruit growth stage, RT fruits wilted and their color became purple brown after freezing, and these brown fruits were counted as freezing samples. Totally, the freezing rate of YL fruits was 8.60%, which was extremely lower than that of RT fruits (55.20%) after freezing damage, indicating that YL fruits were more resistant to cold than RT fruits (Fig. 1c and 1e).
In conclusion, YL showed enhanced freezing tolerance than RT. The fruits of YL had the strongest ability against freezing; however, the flowers also had a modest freezing tolerance.
Proline Was Significantly Accumulated In YL Fruits
Proline and ABA accumulation is commonly considered as the main response of plants to defend against freezing damage [31]. A study has shown that plants would proactively decrease the content of GA to promote freezing tolerance [28]. To clarify the main factors behind the enhanced freezing tolerance in YL, the contents of proline, ABA, and GA were quantified.
For proline, only YL fruits showed a significant increase (almost 1.44-fold) after freezing treatment (Fig. 2a). RT fruits showed no difference in proline content after freezing treatment, but the leaves of both YL and RT had an insignificant reduction in proline content. Thus, proline accumulation might be beneficial for enhancing freezing tolerance in YL fruits.
For ABA and GA, only RT showed significant changes. ABA and GA were increased in the leaves of RT, but GA content was decreased in RT fruits (Fig. 2b). In general, increased ABA and decreased GA are beneficial to freezing resistance [27, 28]. In YL leaves and fruits, ABA or GA showed no difference.
YL Had Stronger Actions On Stomatal Closure
Plant stomata are very important for the exchange of water and CO2; thus, they are important structures that protect cells from cold [32]. Here, we observed stomatal structures with a scanning electron microscope. After freezing stress, the stomatal density of RT and YL leaves was obviously decreased, and only YL showed a significant decrease (Fig. 3).
In fruits, a single stoma and total stomatal area of YL fruits were significantly reduced after freezing (Fig. 4). The stomatal area of YL fruits was 1.77 ± 0.12 and 1.16 ± 0.12 µm2 before and after freezing, respectively. The stomatal apertures of YL fruits were obviously smaller than those of RT fruits as the long and short axes were significantly reduced by 0.8-fold. Conversely, RT fruits did not exhibit any significant differences.
Scanning electron microscopy results showed that stomatal closure is probably an important adaptation strategy to enhance freezing tolerance in YL fruits.
Transcriptome analysis revealed that the fruits and flowers of YL applied different freezing resistance systems
In order to clarify the freezing tolerance mechanism of YL, especially its fruits, three freezing tissues were subjected to high-throughput sequencing in order to screen the differentially expressed genes (DEGs) of YL and RT. A total of 268,698 unigenes were obtained with an N50 of 1100 bp, and they were annotated against NR, NT, Uniprot, Kyoto Encyclopedia of Genes and Genomes (KEGG), eggNOG, and Gene Ontology (GO) databases. Their expression levels were calculated by the reads per kilobase million mapped reads (RPKM) method (Figure S2, Tables S1 and S2). Interestingly, the transcription profiles of flowers were quite different between YL and RT, whereas the fruits and leaves exhibited similar expression patterns, suggesting that these differences were caused by the freezing tolerance system (Figure S2e).
Given that all three tissues of YL exhibited strong freezing tolerance to different degrees (fruits > flowers > leaves), integrative analysis of DEGs was helpful to clarify the antifreeze mechanism of YL, especially the fruits. DEGs were selected by DEGSeq2 software (R package, https://bioconductor.org/packages/release/bioc/html/DESeq2.html) with a threshold of |log2ratio(fold change)|≥1 and q < 0.05 (FDR corrected) (Fig. 5, Tables S3–S5). Finally, a total of 807 DEGs were obtained in all tissues from YL and RT, suggesting that they play key roles in freezing tolerance (Fig. 5a). To verify the transcriptome data, 20 genes were selected to test their expression levels in all samples by qRT-PCR. The results were highly consistent with the transcriptome data (Fig. 5d).
According to the enrichment analysis of functional annotation, freezing leaves only enriched “Ribosome” (Fig. 5b). However, aside from “Ribosome”, flowers also mostly applied series basic nutrients and energy metabolisms (“Citrate cycle,” “Glycolysis,” amino acid metabolisms, and “Oxidative phosphorylation”) and “Phagosome,” suggesting that the flowers were able to respond to freezing stress by alternating some pathways of growth and development when the stress was not severe for them (Fig. 5b and 5c).
The situation in the fruits was quite different (Figs. 5 and 6, Table S3). Aside from several crucial nutrient and energy metabolism pathways including the “Citrate cycle,” “Glycolysis,” and “Oxidative phosphorylation,” fruits recruited more signal transduction and defense pathways, such as “Plant–pathogen interaction,” “Plant hormone signal transduction,” and “MAPK signaling pathways—plant” (Figs. 5b and 6d). Most enrichment pathways were downregulated, but approximately 18.13% (33) of significantly different GO terms (182) were upregulated in YL fruits, such as “DNA integration” and “RNA-direct DNA polymerase activity” (Fig. 6c, Tables S6–S8). Moreover, the first 10 upregulated GO terms were mainly related to DNA synthesis and modification.
Taken together, these results showed that different tissues of YL applied different freezing resistant systems due to unknown reasons. This might explain why different phenotypes were observed in different tissues after freezing treatment.
Neither ABA biosynthesis nor ABA pathway might be the main reason for the freezing resistant
According to our results, the main antifreeze hormone, ABA, was not significantly accumulated (Fig. 2). Although the “Plant hormone transduction” pathway including the ABA signaling pathway significantly differentially expressed and enriched, ABA biosynthesis and signaling transduction pathway seemed not to be the main reason for freezing resistant of YL fruits (Figs. 5 and 6).
According to RNA-seq data, NCED (9-cis-epoxycarotenoid dioxygenase), a key biosynthesis gene of ABA, expressed no difference in freezing YL fruits than RT fruits (Table 1). Since the content of ABA showed no difference, suggesting that accumulation of ABA was not prior to enhance freezing resistant.
Table 1
Expression profiles of several genes involved in freezing resistant in YL loquat
Gene
|
unigenenID
|
Mean_RPKM
|
UP/DOWN
|
q-value
|
Significant
|
YL
|
RT
|
CYP707A
|
TRINITY_DN80955_c0_g1
|
11.81027
|
13.62596
|
DOWN
|
1.016E-05
|
No
|
TRINITY_DN84841_c0_g4
|
3.648425
|
2.456353
|
UP
|
0.0018865
|
No
|
TRINITY_DN83558_c3_g1
|
79.87738
|
139.4272
|
DOWN
|
1.14E-162
|
No
|
TRINITY_DN40284_c0_g1
|
21.7481
|
8.124339
|
UP
|
1.648E-69
|
Yes
|
NCED
|
TRINITY_DN81603_c0_g4
|
145.6801
|
78.82272
|
UP
|
2.76E-192
|
No
|
TRINITY_DN83631_c1_g2
|
1313.666667
|
1733.333333
|
DOWN
|
0.1777836
|
No
|
PP2C
|
TRINITY_DN80306_c1_g1
|
0.07012
|
0.231449
|
DOWN
|
0.0301922
|
Yes
|
TRINITY_DN79313_c3_g4
|
3.342301
|
6.452799
|
DOWN
|
1.22E-09
|
Yes
|
TRINITY_DN87303_c2_g4
|
1.712378
|
3.609193
|
DOWN
|
1.21E-06
|
Yes
|
TRINITY_DN80839_c0_g2
|
4.402092
|
8.070845
|
DOWN
|
5.45E-14
|
Yes
|
TRINITY_DN74622_c5_g4
|
1.259298
|
3.236222
|
DOWN
|
2.26E-12
|
Yes
|
TRINITY_DN77922_c2_g7
|
21.89618
|
52.17555
|
DOWN
|
1.23E-189
|
Yes
|
TRINITY_DN74622_c5_g2
|
1.211203
|
3.299656
|
DOWN
|
3.74E-13
|
Yes
|
TRINITY_DN87297_c1_g1
|
3.307331
|
8.477507
|
DOWN
|
8.68E-18
|
Yes
|
TRINITY_DN83560_c0_g1
|
49.10402
|
123.4612
|
DOWN
|
0
|
Yes
|
TRINITY_DN73587_c0_g1
|
0
|
0.135885
|
DOWN
|
0.0170134
|
Yes
|
TRINITY_DN75528_c1_g2
|
0.095929
|
0.35284
|
DOWN
|
0.0023364
|
Yes
|
TRINITY_DN67835_c0_g2
|
0
|
0.33138
|
DOWN
|
0.0127325
|
Yes
|
TRINITY_DN66956_c0_g5
|
0.082891
|
0.587606
|
DOWN
|
8.99E-09
|
Yes
|
TRINITY_DN71331_c0_g1
|
0.038016
|
0.203548
|
DOWN
|
0.0184379
|
Yes
|
TRINITY_DN83305_c1_g1
|
21.49326
|
59.31025
|
DOWN
|
0
|
Yes
|
SnRK2
|
TRINITY_DN83302_c0_g2
|
1.456622
|
0.65582
|
UP
|
0.0262294
|
Yes
|
SLAC1
|
TRINITY_DN80888_c1_g1
|
6.169313
|
5.061007
|
UP
|
0.1347991
|
No
|
TRINITY_DN84572_c1_g4
|
18.25298
|
16.18598
|
UP
|
0.7817525
|
No
|
KAT1
|
TRINITY_DN86285_c1_g1
|
8.123739
|
9.823269
|
DOWN
|
1.934E-11
|
No
|
CBF
|
TRINITY_DN84340_c0_g1
|
44.02247
|
89.47271
|
DOWN
|
0
|
Yes
|
TRINITY_DN80208_c2_g1
|
125.1356
|
153.6936
|
DOWN
|
4.713E-45
|
No
|
TRINITY_DN82463_c0_g1
|
2.051787
|
16.3135
|
DOWN
|
1.04E-114
|
Yes
|
TRINITY_DN85002_c0_g1
|
0.872187
|
4.385313
|
DOWN
|
7.051E-34
|
Yes
|
TRINITY_DN74667_c1_g2
|
0.724052
|
3.876971
|
DOWN
|
1.067E-35
|
Yes
|
LEA76
|
TRINITY_DN61955_c0_g1
|
235.3333333
|
256
|
DOWN
|
0.097894034
|
No
|
KIN1
|
TRINITY_DN85365_c0_g1
|
6262.333333
|
6141
|
UP
|
9.05E-06
|
No
|
EjDHNs
|
TRINITY_DN80903_c0_g1
|
2176.345
|
3946.551
|
DOWN
|
3.49E-262
|
Yes
|
TRINITY_DN82302_c1_g2
|
6929.04
|
8093.15
|
DOWN
|
1.247E-23
|
No
|
TRINITY_DN77869_c0_g1
|
7532.838
|
9839.322
|
DOWN
|
1.973E-11
|
No
|
TRINITY_DN82302_c1_g1
|
946.9884
|
1524.798
|
DOWN
|
1.97E-154
|
No
|
TRINITY_DN83533_c2_g1
|
7082.951
|
9929.518
|
DOWN
|
1.74E-116
|
No
|
TRINITY_DN80903_c9_g1
|
368.3558
|
596.0102
|
DOWN
|
1.18E-146
|
No
|
TRINITY_DN81442_c0_g1
|
6.478984
|
5.7688
|
UP
|
0.9676955
|
No
|
TRINITY_DN82302_c1_g3
|
1126.443
|
1754.229
|
DOWN
|
2.86E-145
|
No
|
TRINITY_DN80847_c0_g1
|
11.75012
|
18.10928
|
DOWN
|
5.763E-17
|
No
|
TRINITY_DN73189_c0_g1
|
0.564553
|
1.504637
|
DOWN
|
0.0005633
|
Yes
|
TRINITY_DN85489_c5_g1
|
1717.351
|
1894.503
|
DOWN
|
3.619E-44
|
No
|
TRINITY_DN79081_c0_g1
|
1147.612
|
1472.957
|
DOWN
|
3.796E-11
|
No
|
TRINITY_DN84353_c0_g2
|
0.077709
|
0.118863
|
DOWN
|
0.7136006
|
No
|
Furthermore, genes of ABA pathway were investigated. Only two genes, PP2C (protein phosphatase 2C) and SnRK2 (SNF1-related protein kinase 2), which play decisive roles in ABA signal transduction, were all differentially expressed (Table 1). PP2C is a negative regulator in ABA signal transduction [33]. A total of 15 PP2C genes were significantly differentially expressed, and they were all downregulated in freezing YL fruits compared with freezing RT fruits (Table 1). Contrary to PP2C, SnRK2 is a positive regulator but is inhibited by PP2C in ABA signal transduction [33]. According to the transcriptome data, only one SnRK2-like (TRINITY_DN83302_c0_g2, closest to SnRK2.8) was significantly differentially expressed, and it was upregulated by 2-fold in YL fruits exposed to cold stress (Table 1). But SnRK2.6 was the main regulator to promote stomatal closure by increasing the phosphorylation of SLAC1 (slow anion channel 1) and repressing the phosphorylation of KAT1 (potassium channel protein) [34]. In YL fruits, the expression levels of representative SLAC1 (TRINITY_DN80888_c1_g1, TRINITY_DN84572_c1_g4) and KAT1 genes were changed to some degree but not significant (Table 1).
Moreover, we investigated the marker genes of ABA pathway and CBF pathway, and only CBF4 (TRINITY_DN84340_c0_g1) down-regulated for 0.44-times significantly in YL fruits, whereas there was no difference of Rd29b (TRINITY_DN83533_c2_g1 and TRINITY_DN73189_c0_g1), KIN1 (TRINITY_DN85365_c0_g1) and LEA76 (TRINITY_DN75564_c0_g1 and TRINITY_DN61955_c0_g1) between two freezing fruits (Table 1). Due to genomic loss of loquat, RAB19 and COR15B orthologs were not found in these samples. Thus, it seemed that ABA pathway was not the regulation system of YL fruits to promote stomatal closure.
Above all, neither ABA biosynthesis nor ABA pathway might be the main reason for the freezing resistant.