Abstract

   The physiological mechanisms by which imbibed seeds survive freezing
   temperatures in their natural environment have been categorized as
   freezing avoidance by supercooling and freezing tolerance by
   extracellular freeze-desiccation, but the biochemical and molecular
   mechanisms conferring seed freezing tolerance is unexplored. In this
   study, using imbibed Lactuca sativa seeds we show that fast cooled
   seeds (60 °C h^−1) suffered significantly higher membrane damage at
   temperature between −20 °C and −10 °C than slow cooled (3 °Ch^−1) seeds
   (P < 0.05), presumably explaining viability loss during fast cooling
   when temperature approaches −20 °C. Total soluble sugars increase in
   low temperature environment, but did not differ significantly between
   two cooling rates (P > 0.05). However, both SOD activity and
   accumulation of free proline were induced significantly after slow
   cooling to −20 °C compared with fast cooling. RNA-seq demonstrated that
   multiple pathways were differentially regulated between slow and fast
   cooling. Real-time verification of some differentially expressed genes
   (DEGs) revealed that fast cooling caused mRNA level changes of plant
   hormone and ubiquitionation pathways at higher sub-zero temperature,
   whilst slow cooling caused mRNA level change of those pathways at lower
   sub-zero ttemperatures. Thus, we conclude that imbibed seed tolerate
   low temperature not only by physiological mechanisms but also by
   biochemical and molecular changes.
     __________________________________________________________________

   Temperature is a major environmental sieve selecting plant distribution
   in temperate and cold climates[40]^1. In many complex environments,
   plants have to cope with temperatures plummeting as low as −30 °C
   during winter[41]^2. However, most species adapted to these locations
   have evolved with a suite of protective mechanisms, which are mostly
   triggered during a brief exposure to low but non-freezing temperature,
   in light of preparing the plants to further lower temperature, a
   process termed as ‘cold acclimation’[42]^3. Understanding the
   mechanisms of freezing tolerance in plants is of extensive ecological
   and practical importance. Not only do such studies inform us about the
   coping strategies plants use to survive freezing in natural conditions,
   but they also offer great insights to improve the freezing tolerance of
   many agronomic plants that are susceptible to freezing damage.

   Research into identifying the mechanisms by which plants survive low
   temperature has established a multitude of molecular, biochemical, and
   physiological changes are involved in making a plant freezing tolerant.
   One premise is that an array of genes is either up- or down-regulated
   in response to low temperature[43]^4,[44]^5,[45]^6. These waves of
   changes at the transcript levels expressing or suppressing the genes
   presumably encompass numerous functions including transcription,
   signalling, cell wall biogenesis, and synthesis of hormones[46]^4.
   Another line of evidence shows that numerous phytohormones including
   abscisic acid (ABA), auxin, ethylene and cytokinin play a crucial role
   in regulating freezing tolerance processes. Whereas the level of ABA in
   plants increases in the low temperature environment[47]^7, auxin[48]^8,
   cytokinin and ethylene[49]^9 are mostly down regulated in many species.
   These changes in phytohormone levels alter gene expression by inducing
   or preventing the degradation of transcriptional regulators via the
   ubiquitin-proteasome system[50]^10.

   In addition, the levels of soluble carbohydrate in many plant tissues
   are known to increase during low temperature[51]^11. These compounds
   are proposed to serve as cryoprotectants and prevent the freezable
   water present in cells undergoing a phase change[52]^12. Generally,
   water present in the biological samples forms ice when exposed to low
   temperature and more than the traceable quantity of ice in the
   intracellular region is mostly lethal. However, plants mitigate this
   stress by either holding the water in a supercooled state or limiting
   the freezing to extracellular spaces and prevent intracellular ice
   formation (IIF)[53]^13. Thus, successful survival of plant organs
   depends invariably on the ability to avoid IIF.

   One of the widely held assumptions is that plants adapted to a
   cold-environment escape the mortality associated risks of a generative
   phase by completing the reproduction in summer and shed the seeds
   before the onset of winter. These seeds subsequently establish soil
   seed banks and over-winter under dense snow cover before beginning to
   germinate the next summer[54]^14,[55]^15. Because snow-cover provides a
   benign environment to the over-wintering seeds, the risk of mortality
   resulting from ice formation as a consequence of low temperature is
   minimized[56]^16. Therefore, significant attention has been paid to
   understand the survival strategies of individual plant parts
   overwintering above the snow cover, e.g. buds, bark or
   leaves[57]^17,[58]^18. Thus, information on seed survival strategies in
   a low temperature environment is scant, despite some interest in
   understanding the tolerance mechanisms of seedlings, mainly due to the
   assertion that newly germinated seedlings are susceptible to freezing
   injury in exposed soil surfaces. However, at the time of snow-melt in
   late winter, seeds in the soil seed banks are hydrated by the melting
   snow and subsequently exposed to nocturnal temperature plummeting to
   sub-zero levels. Such events are expected to occur more commonly in the
   future due to the effects of global warming reducing the duration of
   snow cover, thereby leaving the seeds exposed to direct low temperature
   late in winter.

   The physiological mechanisms, by which the seeds survive freezing
   stress have been studied in a few species including Arabidopsis
   thaliana[59]^19, Brassica napus cv. Quest[60]^20, Eurotia lanata
   (Pursh) Moq[61]^21, Lactuca sativa[62]^22,[63]^23,[64]^24, Pinus
   sylvestris[65]^25 and Pyrus malus[66]^26,[67]^27. These studies show
   that imbibed seeds survive low temperature either by freezing avoidance
   by supercooling or freezing tolerance by extracellular-freezing. In
   Lactuca sativa, there is evidence to show that the selection of a
   particular mechanism is related to the rate at which seeds are cooled.
   At an environmental cooling rate of <4 °C h^−1, the seeds survived low
   temperature by freezing tolerance, whereas at faster cooling rates the
   seeds undergo supercooling[68]^23. However, the putative biochemical
   and molecular changes in seeds during low temperature survival in the
   imbibed state remain largely superficial, promoting the need for
   further investigation.

   Here we investigate the biochemical and molecular changes that occur in
   imbibed seeds when exposed to low temperature. Lactuca sativa has been
   extensively used as a model species to advance our understanding on the
   physiological mechanisms operating in imbibed
   seeds[69]^22,[70]^23,[71]^24. Therefore, we believe this species might
   be a better candidate for identifying the biochemical and molecular
   mechanisms. Our specific objectives in this study with imbibed L.
   sativa seeds were (1) to revisit the physiological mechanisms at
   different cooling rates; (2) to characterize the biochemical change of
   imbibed freezing seeds at empirical and ecologically meaningful cooling
   rates; and (3) to determine the differential molecular changes
   occurring in seeds under different cooling rates.

Results

Seed imbibition and germination

   The rate at which moisture of the L. sativa seeds incubated at 21 °C
   increased is shown in [72]Fig. 1. The pattern of water absorption is
   tri-phasic. The seeds absorb water rapidly when they come in contact
   with water. However, this rapid uptake of water only lasts for some
   time (from few minutes to few hours depending upon the species). This
   is usually followed by a slow water uptake phase, loosely termed as
   lag-phase, where the seeds do not absorb much water, rather use this
   time to move the hydrated reserved and begin the reactions at molecular
   levels that require water. Once this phase is completed the radicle
   begins to emerge, where additional water is absorbed. Seeds of lettuce
   used in this study began to germinate after 14 h and therefore seeds
   residing above agar at 21 °C for 12 h were considered as fully imbibed
   ([73]Fig. 1). One hundred percent of the seeds germinated within 5
   days, indicating the high viability of the seed lot.

Figure 1. Percentage of water imbibed by Lactuca sativa cv. Iceberg seeds
when imbibed above moist filter paper in 9 mm diameter Petri plates.

   Figure 1
   [74]Open in a new tab

   Seeds at 0 h are from 15% RH and 15 °C.

Freezing treatment and viability assessment

   Germination of fully imbibed seeds declined with decreasing
   temperature. Seeds cooled at both cooling rates survived to a higher
   percentage above −15 °C ([75]Fig. 2). However, none of the fast cooled
   seeds survived at −20 °C, compared with 54% survival of the slowly
   cooled seeds that germinated to normal seedlings when retrieved at
   −20 °C ([76]Fig. 2).

Figure 2. Germination percentage of Lactuca sativa seeds following cooling at
60 °Ch^−1 (fast cooled) and 3 °Ch^−1 (slow cooled) to various sub-zero
temperatures.

   Figure 2
   [77]Open in a new tab

   No significant difference in germination percentage was observed until
   the temperature dropped to −15 °C at both cooling rate. Seeds cooled to
   −20 °C at a faster rate showed no survival but slow cooled seeds
   resulted in significantly higher germination percentage (indicated with
   lower-case alphabet).

Electrolyte leakage analysis

   No significant difference in electrolyte leakage was found between
   control and seeds cooled at either cooling rate above −15 °C (P > 0.05;
   [78]Fig. 3). However, seeds cooled at the faster rate to −20 °C showed
   significantly higher leakage than control seeds and slow cooled seeds
   (P < 0.05). The difference in electrolyte leakage between fast and slow
   cooled seeds became apparent as high as −10 °C ([79]Fig. 3). Overall,
   the results indicate that fast cooled seeds suffered significantly
   higher membrane damage at temperatures between −15 and −20 °C ([80]Fig.
   3).

Figure 3. The electrolyte leakage analysis of imbibed seeds without freezing
(control) and after cooled −20 °C at 3 °Ch^−1 (slow cooling) or 60 °Ch^−1
(fast cooling).

   Figure 3
   [81]Open in a new tab

   Different upper case letters indicate statistically significant
   difference between two different cooling rates at a particular
   low-temperature. Different lower case letters indicate significant
   difference at different low temperatures.

Superoxide Dismutase (SOD) activity was upregulated by slow cooling treatment
at −20 °C

   Imbibed seeds slowly cooled to −20 °C had significantly higher SOD
   activity (521.60 U/g) than both control (355.54 U/g) and fast cooled
   samples (360.15 U/g) ([82]Fig. 4a). However, SOD activity difference
   was not significant between fast cooled seeds retrieved at −20 °C and
   control (P > 0.05).

Figure 4. The physiological changes in process of programmed cooling
treatment with fast and slow cooling rates.

   Figure 4
   [83]Open in a new tab

   (a) SOD activity in seeds imbibed for 12 hours without freezing
   (control) and after cooled to −20 °C at 3 °Ch^−1 (slow cooling) or
   60 °Ch^−1 (fast cooling). (b) The relative accumulation level of free
   proline in seeds imbibed for 12 hours without freezing (control) and
   after cooled to −20 °C at 3 °Ch^−1 (slow cooling) or 60 °Ch^−1 (fast
   cooling). (c) Soluble sugar content measured in seeds imbibed for
   12 hours without freezing (control) and after cooled to −20 °C at
   3 °Ch^−1 (slow cooling) or 60 °Ch^−1 (fast cooling). Statistically
   significant difference between groups are marked with different
   alphabets.

Slow cooling caused higher accumulation of free proline at −20 °C

   The free proline based on UV absorbance in seeds fast cooled to −20 °C
   did not differ significantly to the seeds prior to cooling (i.e.
   control) (3.59 ug/g FW vs 2.16 ug/g FW, P > 0.05, [84]Fig. 4b). But a
   significant induction of free proline was observed in the seeds cooled
   slowly to −20 °C (4.60 ug/g FW) when compared with control seeds
   (P < 0.05) ([85]Fig. 4b).

Soluble sugar content is not related to the freezing tolerance developed in
slow cooling treatment

   The level of soluble sugar was increased in seeds exposed to low
   temperature compared with control ([86]Fig. 4c). Although the seeds
   cooled at 60 °Ch^−1 resulted in more levels of sucrose (2.93 mg/g FW)
   compared with seeds cooled at 3 °C h^−1 (2.77 mg/g FW), there was no
   statistically significant difference of sucrose content recorded
   between two cooling rates (P > 0.05).

Auxin and ABA expressed significantly higher in slow cooled seeds

   Under control condition, the content of ABA was 0.159 ± 0.078 ug g^−1
   FW, and the IAA content was 0.125 ± 0.012 ug g^−1 FW. However, both
   fast and slow cooling led to significantly higher accumulation of ABA
   and IAA (P < 0.05). In particular, seeds cooled slowly to −20 °C
   resulted in significantly higher level of ABA (1.033 ± 0.043 ug g^−1 FW
   for fast cooling vs. 1.406 ± 0.13 ug g^−1 FW for slow cooling;
   P < 0.05) and IAA (0.256 ± 0.015 ug g^−1 FW for fast cooling vs.
   0.477 ± 0.027 ug g^−1 FW for slow cooling, P < 0.05).

RNA-seq analysis

   RNA-seq analysis conducted on fully imbibed seeds cooled to −20 °C at
   two different cooling rates showed that an average of 12,000,000 reads
   were obtained in each sample ([87]Table 1). We were able to map 65~67%
   of the total reads to the genome sequences with more than 65% of the
   total reads uniquely matching and the multi-position matched reads
   occupied no more than 0.67% ([88]Table 1).

Table 1. Statistics of RNA-seq analysis.

   Sample ID Control Fast cooling Slow cooling
   Total reads 12,413,353 (100.00%) 11,702,598 (100.00%) 11,527,110
   (100.00%)
   Total Base Pairs 608,254,297 (100.00%) 573,427,302 (100.00%)
   564,828,390 (100.00%)
   Total Mapped Reads 8,429,247 (67.90%) 7,730,392 (66.06%) 7,579,398
   (65.75%)
   Perfect Match 6,873,209 (55.37%) 6,296,355 (53.80%) 6,051,017 (52.49%)
   <=2 bp Mismatch 1,556,038 (12.54%) 1,434,037 (12.25%) 1,528,381
   (13.26%)
   unique match 8,350,513 (67.27%) 7,651,551 (65.38%) 7,505,842 (65.11%)
   Multi-position Match 78,734 (0.63%) 78,841 (0.67%) 73,556 (0.64%)
   Total Unmapped Reads 3,984,106 (32.10%) 3,972,206 (33.94%) 3,947,712
   (34.25%)
   [89]Open in a new tab

   The number of differentially expressed genes (DEGs) varied
   significantly between the treatments ([90]Fig. 5). There were 1721 DEGs
   between control and fast cooling treatment, in which 968 were
   upregulated and 753 were down-regulated. A total of 1987 DEGs were
   observed between control and slow cooling treatment in which 1089 were
   upregulated and 898 were down-regulated. However, out of 1735 DEGs
   between fast and slow cooling treatment, 780 were identified to be
   upregulated and 955 were down-regulated. Notably, there were 570 DEGs
   in both fast and slow cooling when compared with control treatment,
   indicating these were cold responsive genes. Of these 570 DEGs, 11
   genes including leucine-rich repeat extensin-like protein 4-like and
   clock regulator Time For Coffee, etc were up-regulated during fast
   cooling but down-regulated during slow cooling. By contrast, lanC-like
   protein-2 like down-regulated during fast cooling was up-regulated when
   seeds were cooled slowly. All the 12 genes expressed in opposite
   directions between fast and slow cooling are listed in [91]Table 2.

Figure 5. Number of differentially expressed genes (DEGs) amongst control,
fast and slow cooling treatment.

   Figure 5
   [92]Open in a new tab

Table 2. The DEGs with up or down regulated between fast and slow cooling
treatment compared with control.

   GeneID Blast nr log2 Ratio(F/C) Up-Down FDR log2 Ratio(S/C) Up-Down FDR
   gi|90503171 lanC-like protein 2-like −1.12 Down 3.45E-6 1.05 Up
   5.43E−11
   gi|22259961 leucine-rich repeat extensin-like protein 4-like 1.65 Up
   4.53E-11 −2.01 Down 1.16E-4
   gi|22250752 Clock regulated protein, TIME FOR COFFEE-like 1.49 Up
   6.90E-57 −2.33 Down 2.18E-35
   gi|22225830 − 1.44 Up 2.04E-17 −1.69 Down 6.08E-08
   gi|22250698 − 1.41 Up 3.40E-28 −1.66 Down 5.37E-13
   gi|22240524 − 1.36 Up 1.15E-13 −1.04 Down 0.000988
   gi|22265151 unnamed protein product 1.33 Up 2.23E-18 −1.13 Down
   9.17E-06
   gi|22253147 DEAD-box ATP-dependent RNA helicase 42-like 1.32 Up
   1.08E-28 −1.12 Down 1.35E-08
   gi|22242137 unnamed protein product 1.16 Up 6.30E-13 −1.27 Down
   1.14E-06
   gi|22254644 glutamate synthase, putative (NADH dependent) 1.10 Up
   4.62E-08 −1.34 Down 3.78E-05
   gi|90517427 Unnamed protein 1.06 Up 9.68E-31 −1.40 Down 7.55E-22
   gi|83979007 glutamate synthase, putative 1.04 Up 5.75E-18 −1.03 Down
   2.19E-08
   [93]Open in a new tab

Pathway enrichment

   There were 1027 DEGs that can be annotated to KEGG pathways between
   control and slow cooling treatment; 904 between control and fast
   cooling treatment; 789 between fast and slow cooling treatment. Based
   on the pathway enrichment results using DEGs, the significant pathways
   between control, fast and slow cooled treatments can be classified into
   4 types ([94]Table 3). Type I is the pathways that differed
   significantly only in slow cooling based on the comparison with
   control, and include RNA transport, spliceosome, plant hormone signal,
   endocytosis, pyrimidine metabolism, and nucleotide excision repair.
   Type II encompasses pathways that were induced in both cooling
   treatments but enriched more significantly in slow cooling treatment
   than fast cooling, and include homologous recombination, DNA
   replication, and ubiquitin mediated proteolysis. Type III contains
   pathways that differed significantly only in fast cooling based on the
   comparison with control and include alanine, aspartate and glutamate
   metabolism and fatty acid elongation. Type IV contains pathways that
   were induced with no difference between the two cooling rates. This
   type includes protein processing in the endoplasmic reticulum and plant
   circadian rhythm. The pathways included in type IV may be the common
   pathways of both fast and slow cooling treatment.

Table 3. The significantly enriched pathways in fast and slow cooling
treatment.

   Types Pathways ^aP-value(fast/slow) ^bP-value (control/fast)
   ^cP-value/(control/slow) ID
   I RNA transport 2.95e–05 — 39 (0.38%)/0.001 ko03013
   Spliceosome 29 (3.68%)/0.001 — 32 (3.12%)/0.008 ko03040
   Plant hormone signal transduction 44 (5.58%)/0.001 — 55 (5.36%) 7.79–e4
   ko04075
   Endocytosis 15 (1.9%)/0.032 — 18 (1.75%)/0.040 ko04144
   Pyrimidine metabolism 16 (2.03%)/0.041 — 20 (1.95%)/0.035 ko00240
   Nucleotide excision repair 8 (1.01%)/0.055 — 14 (1.36%)/0.001 Ko03420
   II Homologous recombination 6 (0.76%)/0.006 6 (0.66%)/0.011 6
   (0.58%)/0.019 ko03440
   DNA replication 7 (0.89%)/0.013 7 (0.77%)/0.026 14 (1.36%)/5.53e–6
   ko03030
   Ubiquitin mediated proteolysis 18 (2.28%)/0.064 23 (2.54%)/0.014 29
   (2.82%)/0.001 ko04120
   III Alanine, aspartate and glutamate metabolism 15 (1.9%)/0.004 14
   (1.55%)/0.025 14 (1.36%)/0.061 ko00250
   Fatty acid elongation 6 (0.76%)/0.063 8 (0.88%)/0.016 — ko00062
   IV Protein processing in endoplasmic iculum — 41 (4.54%)/0.002 40
   (3.89%)/0.021 ko04141
   Circadian rhythm - plant — 12 (1.33%)/0.021 13 (1.27%)/0.024 ko04712
   [95]Open in a new tab

   Note: ^aPathway enriched using DEGs between fast and slow cooling
   treatment; ^bPathway enriched using DEGs between control and fast
   cooling treatment; ^cPathway enriched using DEGs between control and
   slow cooling treatment. I. Significant in slow cooling treatment; II.
   More significant in slow cooling than in fast cooling; III. More
   significant in fast cooling treatment; IV. Significant in both cooling
   treatments and no significant difference between the two kinds of
   cooling rates. Note: −P > 0.05.

Verification of factors in plant hormone and ubiquitin signal pathways by
real time PCR

   Pathway enrichment results indicated that both plant hormone and
   ubiquitin E3 ligase mediated proteolysis were modified during slow
   cooling treatment compared with fast cooled seeds ([96]Fig. 6).
   According to RNA-seq, after cooling the imbibed seeds at 3 °Ch^−1 to
   −20 °C, the transcription of ARF1 was induced, while TIR1 was reduced
   in fast-cooled seeds. In addition, the transcription of ethylene
   pathway factors including EBF and EIN2 were reduced, but the ABA
   pathway gene ABF was induced at −20 °C ([97]Fig. 6).

Figure 6.

   [98]Figure 6
   [99]Open in a new tab

   The mRNA level change of factors in plant hormone pathway (A) and
   ubiquitinn E3 ligase (B) between fast and slow freezing based on
   RNA-seq[100]^59. The red rectangles represent up-regulation and green
   rectangles represent down-regulation of genes in slow freezing compared
   with fast freezing.

   We performed real-time PCR to specifically characterize the temperature
   at which the mRNA level changes for factors of these two pathways by
   determining the changes after cooling the seeds to −5, −10, −15 and
   −20 °C ([101]Figs 7 and [102]8). During slow cooling, the mRNA level
   change of above mentioned factors occurred at −20 °C, and no
   significant change was evident at −5, −10 and −15 °C ([103]Fig. 7).
   However, during fast cooling treatment, although there was no
   significant mRNA level change for above plant hormone pathway factors
   at −20 °C, the mRNA level change of auxin and ethylene pathway related
   factors including ARF1, TIR1, EIN2, EBF occurred at temperatures as
   above at −10 °C. Nevertheless, this increased mRNA level went back to
   original level at −15 °C at fast cooling treatment ([104]Fig. 7,
   [105]Table 4). In contrast, the mRNA level change of ABF occurred at
   −5 °C during fast cooling which indicates the faster response of ABA to
   cooling than auxin and ethylene signals ([106]Table 4). However, the
   mRNA level of above factors gradually went back to the original level
   in the seeds cooled to −15 °C at a fast rate. Although the temperature
   at which the mRNA level changed varied between cooling rates, the
   tendency to increase or decrease was the same at both cooling rates.
   For example, ARF1 and ABF showed positive response while TIR1, EIN2 and
   EBF showed negative response under both cooling treatments ([107]Fig.
   7, [108]Table 4). Moreover, the two auxin-related factors exhibited
   antagonistic expression in seeds, while EIN2 and EBF exhibited
   synergistic expression ([109]Fig. 7, [110]Table 4).

Figure 7.

   [111]Figure 7
   [112]Open in a new tab

   The verification of RNA-seq for genes in plant hormone pathways by real
   time PCR, (a) EIN2; (b) EBF; (c) TIR1; (d) ARF1 and (e) ABF. Different
   lower case letters indicate the significant difference between
   temperatures. Different higher case letters indicate the significant
   difference between cooling rates.

Figure 8.

   [113]Figure 8
   [114]Open in a new tab

   Verification of RNA-seq for factors in SCF complex by real time PCR,
   (a) F-box and (b) SKP 1. Different lower case letters indicate the
   significant difference between temperatures. Different higher case
   letters indicate the significant difference between cooling rates.

Table 4. The response of plant hormone pathway genes to fast and slow
cooling.

   Temperature
     __________________________________________________________________

   Fast cooling
     __________________________________________________________________

   Slow cooling
     __________________________________________________________________

   Response to cold Expression correlation of detected genes in single
   pathway
   Pathways Genes −5 °C −10 °C −15 °C −20 °C −5 °C −10 °C −15 °C −20 °C^*
   Auxin related ARF1 0 +++^* + − + 0 0 +^* Positive Antagonistic
   TIR1 0 —* 0 0 0 0 0 —* Negative
   Ethylene related EBF 0 —* 0 − 0 0 + —* Negative Synergestic
   EIN2 0 —^* 0 0 0 0 + —* Negative
   ABA related ABF +++^* ++^* + 0 0 0 0 ++* Positive —
   Ubiquitin related Skp1 0 0 +++^* − 0 0 + +++* Positive Synergestic
   F-box 0 +++^* 0 0 0 0 0 +++* Positive
   [115]Open in a new tab

   Note: *indicated that the response occurred at the early stage of fast
   cooling or later stage of slow cooling. “+” indicated up-regulation and
   “−” indicated down-regulation. The number of “+” or “− ”indicated the
   significance of the differential expression. If the number was 1,
   0.001 < P < 0.05. If the number was 2, 0.0001 < P < 0.001. If the
   number was 3, P < 0.0001, “0” indicated that P > 0.05.

   The ubiquitin mediated proteolysis pathway (enriched) was significantly
   different between slow cooling and fast cooling treatments ([116]Table
   3). Since SCF has been proven to be involved in plant hormone signal
   pathways, we detected the duration change of SCF factors including Skp1
   and F-BOX ([117]Fig. 8). Both Skp1 and F-BOX were induced by slow
   cooling treatment according to RNA-seq. The real time PCR verification
   was consistent with the RNA-seq results ([118]Fig. 6). The peak level
   of Skp1 existed at −15 °C during fast cooling and −20 °C when the seeds
   were cooled at the slower rate of 3 °Ch^−1. The peak level of F-BOX
   existed at −10 °C and −20 °C in fast and slow cooled seeds,
   respectively. Similar to the factors of plant hormone signal pathway in
   fast cooling treatment, the expression levels of these factors went
   back to the original level at later stage(s) ([119]Fig. 8, [120]Table
   4).

Discussion

   A combination of physiological and molecular events occurred in imbibed
   L. sativa seeds when exposed to low temperature. Based upon freezing
   test results, it appears that iceberg lettuce seeds undergo
   supercooling at the faster cooling rate, but at a slower or more
   ecologically meaningful cooling rate the seeds develop freezing
   tolerance by extracellular freeze induced desiccation as a survival
   mechanism contributing to survival below −15 °C ([121]Fig. 1). This
   finding is in accordance with the previous results on several cultivars
   of lettuce as proposed by Keefe and Moore[122]^23. However, this
   pattern of survival strategy is not universal in seeds because some
   cultivators of lettuce did not survive −20 °C regardless of cooling
   rate[123]^24,[124]^28. Such discrepancy is also found in other seed
   systems tested, e.g. Pinus sylvestris[125]^25. Furthermore, the
   successful survival of iceberg lettuce seeds by extracellular freezing
   has a lower limit of around −40 °C compared with supercooled seeds
   losing viability at temperatures around −18 °C[126]^29.

   From the premise that membrane damage is directly related to the
   morality of seeds at low temperature[127]^30, the results that slow
   cooled seeds show relatively little or no membrane damage compared with
   fast cooled seeds when cooled to −20 °C are of particular interest. The
   increased electrolyte leakage at −20 °C during fast cooling corroborate
   with the mortality of seeds at this temperature suggesting that
   membrane damage occurred possibly due to the formation of ice in
   supercooled water disrupting the membranes. However, various
   biochemical substances produced in seeds are known to protect seeds
   under low temperature environment. Soluble sugars are present in seeds
   of many species have been proposed to play an important role in
   conferring seed survival during stress related to desiccation. When the
   free water present in the seeds is removed, the sugars actively
   participate in maintaining the structural integrity of cells and
   preventing the disruption of cellular structures at a wide-range of
   temperature up to −196 °C. In some woody plants, soluble sugars
   accumulate from autumn to winter which correlated with an increase in
   freezing tolerance[128]^31. Further, it has been reported that
   cold-acclimation results in increased soluble sugar levels,
   specifically raffinose and stachyose, which would subsequently increase
   the freezing tolerance capacity of many plant parts[129]^32. However,
   whether the levels of soluble sugar affect the extent of freezing
   tolerance in seeds could not be determined due to the small increase in
   soluble sugar at both cooling rate ([130]Fig. 3). Alternatively,
   imbibition in seeds may trigger germination events which might increase
   the soluble sugar levels. Thus, the importance of soluble sugars in
   freezing tolerance of seeds still awaits more specified investigation.

   Nevertheless, the increased amount of SOD activity and proline
   accumulation during slow cooling indicate these changes likely play a
   pivotal role in protecting membranes during freezing. The damage of
   membrane integrity at a low temperature environment is attributed to
   the phase transition of membrane fatty acids from liquid-crystalline to
   solid-gel state due to the degradation of lipids by enzymes[131]^33.
   The second set of explanation for membrane damage during low
   temperature is the stress environment produces free radical and
   reactive oxygen species (ROS) which affect cellular functions[132]^34.
   Many studies have proposed that SOD will reduce ROS, thus protect
   membrane during low temperature stress[133]^35. Emerging findings also
   support the importance of free proline in protecting
   membranes[134]^36,[135]^37. Results of this study strongly suggest that
   slow cooling induces higher proline accumulation and SOD activity to
   maintain membrane integrity.

   In the recent years, a great deal of attention has been given to
   identify the genes involved in plant freezing tolerance. In recent
   years, RNA-seq is becoming a revolutionary tool for investigating the
   molecular mechanism in a robust way[136]^38 and this method has been
   successfully applied for seed systems[137]^39. In the present study, we
   used RNA-seq to determine the changes of gene expressions occurring in
   seeds when cooled to −20 °C. In line with the current understanding of
   plants and seedlings, our results show that numerous genes and
   functional pathways were up or down-regulated in imbibed lettuce seeds
   when moved to low temperature conditions from room temperature
   ([138]Fig. 5).

   Approximately 550 genes changed during both fast and slow cooling
   indicating that these genes were expressed as a result of low
   temperature environment. However, 12 of these genes were either
   up-regulated or down-regulated depending on the cooling rate employed.
   In this regard, it is of interest to note that leucine-rich repeat
   extensin-like protein 4-like, clock regulator Time For Coffee and a
   dead-box RNA helicase up-regulated during fast cooling treatment were
   down-regulated when seeds were cooled slowly. Leucine-rich repeat
   extensin-like protein 4 is a protein related to root morphogenesis and
   pollen development[139]^40, as well as the plant -microbe
   interaction[140]^41. However, there has been no report about the
   correlation of this gene with low temperature. The TIME FOR COFFEE
   (TIC) encodes a nuclear regulator in the Arabidopsis thaliana circadian
   clocks[141]^42. TIC acts as a negative factor in the JA-signaling
   pathway which mediates various stress responses[142]^43. This is the
   first study showing the involvement of this gene in cold tolerance, but
   the interaction of circadian rhythm and cold acclimation has been
   reported in voluminous studies[143]^44. Likewise, One DEAD-box
   ATP-dependent RNA helicase was found to be up- and down-regulated
   during fast and slow cooling respectively. The DEAD-box helicase is
   believed to play an important role in cold stress defense in bacteria
   by the unwinding cold-stabilized secondary structure in the 5′-UTR of
   RNA[144]^45. On the other hand, lanC-like protein 2 down-regulated
   during fast cooling was up-regulated during slow cooling. LanC-like
   protein 2 is similar to G protein coupled receptor (GCR) 2-like genes.
   GCR 2 is identified to be the membrane receptor of ABA[145]^46. In
   lettuce, LanC may be the key receptor gene in ABA signal during low
   temperature stress[146]^47.

   Our study also focused on relating these genes to functional pathways
   occurring in imbibed seeds after cooling the seeds at two different
   cooling rates. Based on pathway enrichment of DEGs of control-fast
   cooling treatment, control-slow cooling treatment and fast-slow cooling
   treatment, the significant pathways between fast and slow cooling
   treatment at −20 °C can be classified into 4 types, which included
   pathways enriched in slow cooling or enriched in fast cooling
   treatment, or both ([147]Table 3). It seems that the regulation of
   genetic or epigenetic aspects was changed more after slow cooling
   treatment such as RNA transport, DNA replication and ubiquitin mediated
   proteolysis, while metabolism of small chemicals including alanine,
   aspartate, glutamate metabolism and fatty acid elongation were induced
   more during fast cooling. There are reports about the cold response of
   RNA processing including RNA splicing, export and secondary structure
   unwinding in plants[148]^48 and the induction of Ala, Asp and Glu under
   cold stress[149]^49. However, it remains unknown at this time, whether
   sudden temperature plummetleads to cytoplasmic biochemical change at
   lower sub-zero temperature while the slow cooling leads to gene
   expression change in the nucleus.at the same point.

   Pathway enrichment based on RNA-seq indicated that plant hormones
   including ABA, auxin, and ethylene are believed to be an important
   regulator of cold stress response[150]^8. Ubiquitination also plays a
   significant role in cold acclimation[151]^50. The two factors in auxin
   pathway including ARF1 and TIR1 are the targets of miR393 which is
   involved in cold stress[152]^51. Ethylene pathway factors EBF and EIN2
   are known to involve in fruit ripening disorder caused by chilling
   injury[153]^52. ABF is an important regulator in ABA response pathway.
   ABF/ABA-responsive element binding protein (AREB) are basic region
   leucine zipper (bZIP)-type DNA-binding proteins that regulate the
   expression of Cor (Cold Responsive)/Lea genes that contain cis-acting
   elements ABA-responsive element (ABREs) in their promoters[154]^53.
   F-box and Skp1 are components of E3 ligase. However, there has been no
   report about the involvement of Skp1 and F-box in cold response.
   Nevertheless, based on our RNA-seq results, Skp1 and F-box were
   up-regulated at −20 °C after slow freezing. How Skp1 and F-box are
   involved in freezing tolerance of seeds remains an open area to be
   investigated in the future.

   To date, most of the freezing tolerance studies on physiological and
   molecular mechanisms involved in plants have focused on plant tissues
   and there have been no in-depth studies available on seed systems (see
   Introduction). Whilst, it is clear that the mechanisms involved in
   plants vary with species or plant parts under investigation, several
   new pathways that are identified here to involve in seed freezing
   tolerance[155]^1,[156]^2,[157]^3. A direct comparison of these
   mechanisms with the known information plant systems seems less
   plausible. However, from our results, it appears that there are
   specific changes occurring at molecular level in the seed systems
   ([158]Figs 7 and [159]8). The non-enrichment of plant hormone pathway
   in fast freezing treatment observed by RNA-seq was probably a
   consequence of quick response of seeds to these events. Thus, most of
   the molecular changes occurred at higher sub-zero temperatures in fast
   cooled seeds. Based on this evidence, we suggest that a sudden cold
   shock stimulates all these changes when the temperature around seeds is
   rapidly reduced. By contrast, seeds do not undergo any changes at
   higher sub-zero temperature when the drop in temperature was slow at a
   more ecologically meaningful rate, presumably sensing the temperature
   change is a temporary stress. However, when the temperature was reduced
   to critical levels of −15 °C, a sudden burst of changes occurs
   ([160]Figs 7 and [161]8), which contributes to seed survival at the
   slower cooling rate. Thus, similar to the physiological mechanisms,
   seeds have evolved with specific biochemical and molecular mechanisms
   to protect freezing stress that are specifically affected by cooling
   rate. Nevertheless, the significance of differential timing for
   regulation of above factors at different cooling rates demands special
   emphasis in the future studies.

Methods

Seed materials and germination

   Seeds of Lactuca sativa cv. Iceberg were purchased from a retail outlet
   and stored at 15% RH and 15 °C prior to experimentation. The initial
   germination level was determined by sowing four replicates of 25 seeds
   on 1% agar-water in Petri dishes. The Petri dishes were incubated at
   21 ± 1 °C constant temperature with 16/8 h light/dark regime with a
   light intensity of c. 30 μmol m^−2 s^−1. The seeds were deemed to have
   germinated when the radicle had emerged by 2 mm or more.

Seed imbibition and moisture content

   Ten replicates of 25 seeds were imbibed on 1% agar-water in Petri
   dishes one Petri dish was sampled every 8 hours for moisture content
   determination for 80 h. Moisture content was determined gravimetrically
   by oven drying at 103 °C for 17 h and expressed on a fresh weight basis
   throughout. The time at which the first germination took place was also
   recorded. Whenever, imbibed seeds were used in the experiments, the
   seeds took water for 12 hours (see results).

Freezing tests

   Fully imbibed, but non-germinated seeds (see [162]Fig. 1) were surface
   dried by placing them between soft tissue pads. These seeds were loaded
   in cryovials and cooled in a programmable freezer at 60 °C or 3 °C h^−1
   to the terminal temperature of −20 °C. Viability assessments were made
   by sampling three replicates of 25 seeds at each 5 °C interval and
   germinating them under conditions described above.

The relative electrolyte leakage (REL) assay

   REL analysis was conducted in imbibed seeds with both non-freezing
   treatments and cooled to −5, −10, −15 and −20 °C at fast and slower
   rates using a method described by Chowhan et al.[163]^54. Briefly,
   0.5 g seeds were added into the 50 ml-tubes with 5 mL deionized water,
   then mixed thoroughly by placing the tubes in a shaker (Kylin-Bell Lab
   Instruments Co. Ltd., Jiangsu, China) for 2 h before measuring the
   electrolyte leakage (EL) using a conductivity meter (Cold-Parmer, USA).
   EL was also measured after boiling the tubes with contents for 30 min.
   REL was expressed as the ratio of initial to final conductivity.
   De-ionised water was used as a control. The experiment is replicated
   twice and the values are presented as average.

Detection of soluble sugar changes by HPLC

   Previous studies on lettuce, and data collected in the present
   investigation (see results) indicate that supercooling ability is
   generally lost in the region of −18 °C; therefore, any molecular change
   contributing to the survival of seeds is expected to occur at
   temperatures above −20 °C. Consequently, we hypothesized cooling seeds
   to −20 °C could serve as an appropriate threshold for studying the
   importance of soluble sugar content. To determine the sugar level
   changes, imbibed seeds were cooled separately at 60 °C or 3 °C h^−1 to
   −20 °C in a programmable freezer. These seeds were ground in a mortar
   and pestle in the presence of liquid nitrogen. The powder was
   homogenized by adding an equal volume of 80% ethanol and incubated at
   50 °C for 20 min. The homogenate was then centrifuged for 30 min at
   3000 rpm and the supernatant was collected and the remaining pellets
   were washed with 80% ethanol and re-centrifuged. This process was
   repeated thrice and all the supernatants collected were mixed. The
   ethanol in the supernatant was removed with a rotary vacuum evaporator
   at 50 °C. The remaining liquid was filtered with a 0.45 uM microporous
   filter membrane (Millipore, USA). The filtrate was diluted 10 fold by
   adding ddH[2]O and analyzed by HPLC with a Waters Associates Liquid
   Chromatograph (Milford, MA, USA), equipped with an isocratic1525 pump,
   U6 K injector. Sucrose was detected with a 2414 differential
   refractometer. The immobile phase was A P S- H Y P E R S IL
   (4.6 mm × 250 mm) and the mobile phase was acetonitrile: water (80:20)
   at a flow rate of 0.8 ml/min at room temperature. The volume of
   injection was 10 uL. The standard samples were prepared as follows: the
   sucrose samples were dried at 105 °C for 5 hr and 0.5 g sample was
   dissolved in water and diluted to 1, 2, 5, 10 mg/ml, respectively and
   filtered with a 0.45 uM membrane (Millipore, USA).

Superoxide Dismutase (SOD) assay

   Total SOD activity was measured following the method described by
   according to Beyer and Fridovich[164]^55. The total SOD activity was
   detected in imbibed seeds with non-freezing treatment (control) and
   imbibed seeds cooled to −20 °C under both the cooling rates. The total
   SOD activity was assayed photochemically based on the inhibition of NBT
   reduction by superoxide radicals. One unit of SOD was defined as the
   amount of enzyme required to inhibit the reduction rate of NBT by 50%
   at 25 °C.

Free proline assay

   Free proline was extracted using the method developed by Bates et
   al.[165]^56. Imbibed seeds with non-freezing treatment (using as
   control) and imbibed seeds cooled to −20 °C under both the cooling
   rates were ground and mixed with 5 ml of 3% aqueous sulfosalicyclic
   acid. The mixture was boiled for 10 min and centrifuged at 12000 rpm
   for 10 min. The discarded supernatant was subsequently mixed with 2 ml
   acid-ninhydrin and 2 ml glacial acetic acid. After boiling for 10 min,
   the reaction mixture was extracted with 4 ml toluene by centrifugation
   for 5 min at 3000 rpm. Then the absorbance was measured at 515 nm using
   NANO DROP2000 (Thermo Fisher Scientific, Waltham, MA USA). Standard
   curves were made using 2 ml standard solutions of proline with
   different concentration, prior to sample measurement.

ABA and IAA content assay

   ABA and IAA was extracted by modified method of the previous
   report[166]^57. Briefly, two grams of seeds in fresh weight were
   grounded into powder. Add 20 ml 80% methanol and shaked overnight at
   4 °C. Then the extracting solution was evaporated to a volume of 4 ml
   and extracted by chloroform and ethyl acetate seperately. The
   extracting solution was evaporated to be dry and the extracts was
   dilluted by methanol. ABA was detected by HPLC and IAA was detected by
   LC-MS based on the previous method[167]^57.

Determination of molecular changes in freezing seeds using RNA-seq analysis

   To unravel the molecular changes occurring in seeds during low
   temperature, RNA-seq analysis was performed in BGI company (Shenzhen,
   China) using imbibed seeds (control) and after cooling seeds to −20 °C
   at 60 °C or 3 °C h^−1. For RNA extraction, seeds cooled to −20 °C were
   ground using a mortar and pestle in the presence of liquid nitrogen.
   The samples were first treated with DNase I to degrade any possible DNA
   contamination and the mRNA was enriched by using oligo (dT) magnetic
   beads on the ice. The mRNA was then sliced into short fragments by
   mixing the sample with fragmentation buffer. Using the short fragments,
   we synthesized the first strand of cDNA by using random hexamer-primer.
   Later, Buffer, dNTPs, RNase H and DNA polymerase I were added to
   synthesize the second strands. The double strand cDNA was purified with
   magnetic beads. End reparation and 3′-end single nucleotide A (adenine)
   addition was then performed. Finally, sequencing adaptors were ligated
   to the fragments. The fragments were enriched by PCR amplification.
   During the quality control step, Agilent 2100 Bioanaylzer and ABI
   StepOnePlus Real-Time PCR System were used to qualify and quantify the
   sample library, which was sequenced via Illumina HiSeqTM 2000.

   Data filtering was carried out to obtain high quality reads as the
   clean reads including removing reads with adaptor sequences, removing
   reads in which the percentage of unknown bases (N) was greater than
   10%, and removing low quality reads. If the percentage of the low
   quality base (base with quality value ≤5) was greater than 50% in a
   read, we define this read as low quality. Clean reads were mapped to
   reference sequences and/or reference gene set using
   SOAPaligner/SOAP2[168]^58. No more than 2 mismatches were allowed in
   the alignment. The expression level for each gene was determined by the
   numbers of reads uniquely mapped to the specific gene and the total
   number of uniquely mapped reads in the sample. Briefly, the gene
   expression level was calculated by reads Per kb Million reads (RPKM)
   method.

   The probability of gene A expressed equally between fast and slow
   cooled samples was calculated as [169]equation (1):
   graphic file with name srep44166-m1.jpg

   where, N1 is the number of total clean tags of the fast cooled sample,
   N2 is the number of total clean tags of slowly cooled sample, x is the
   number of tags held by gene A in slowly cooled sample, and y is the
   number of tags held by gene A in the slow cooled sample.

   The calculated P-value corresponds to differential gene expression.
   False discovery rate (FDR) was used to determine the threshold of P
   value in multiple tests. We used “FDR ≤0.001 and the absolute value of
   log2Ratio ≥1” as the threshold to judge the significance of gene
   expression difference.

   To classify the function of differentially expressed genes (DEGs),
   pathway enrichment analysis was performed by mapping the DEGs to KEGG
   database[170]^59, using the formula described by Hooper and
   Brok[171]^60. Briefly, this involves calculating gene numbers for every
   term, then using the hypergeometric test to find significantly enriched
   terms in DEGs. The following [172]equation (2) was used to map the
   DEG’s to KEGG database:
   graphic file with name srep44166-m2.jpg

   where N is the number of all genes with KEGG annotation, n is the
   number of DEGs in N, M is the number of all genes annotated to specific
   pathways, and m is the number of DEGs in M. The calculated P value was
   tested for statistical significance using Bonferroni correction, at
   P = 0.05.

Verification by real time PCR

   The RNAseq displayed a wave of changes occurring at −20 °C in both fast
   and slow cooled seeds (see results). To understand the temperature at
   which these changes occur in imbibed seeds at two different cooling
   rates, we verified the sequencing by extracting total RNA from imbibed
   seeds after cooling to −5, −10, −15, −20 °C at 60 °C or 3 °Ch^−1. The
   samples were ground with a mortar and pestle in the presence of liquid
   nitrogen. The powdered seeds were treated with RNAiso Plus (TaKaRa) and
   cDNA was synthesized using reverse transcriptase (Prime Script RT
   reagent Kit with gDNA Eraser, Takara, Japan). Real time PCR was
   performed with PrimeScript™ RT reagent Kit and gDNA Eraser (Perfect
   Real Time) (Takara, Japan) by ABI7900 system (ABI, USA). The conditions
   were as follows: 1 cycle for degeneration at 94 °C (3 min), 40 cycles
   at 94 °C (20 s), 52 °C (20 s), 72 °C (20 s) for amplification and 1
   cycle for elongation at 72 °C (7 min). Three replicates were carried
   out. The sequences of primers are listed in [173]Supplementary Table 1.

Statistical analysis

   Percentage germination of seeds after cooling at 60 °C and 3 °Ch^−1 to
   specific low temperature was compared using a t-test. The different
   groups of REL, SOD, proline and soluble sugars data of control, fast
   cooled and slow cooled seeds were tested for statistical significance
   by one-way ANOVA independently. We used LSD post-hoc test to find
   significant differences between groups.

Additional Information

   How to cite this article: Jaganathan, G. K. et al. Physiological
   Mechanisms Only Tell Half Story: Multiple Biological Processes are
   involved in Regulating Freezing Tolerance of Imbibed Lactuca sativa
   Seeds. Sci. Rep. 7, 44166; doi: 10.1038/srep44166 (2017).

   Publisher's note: Springer Nature remains neutral with regard to
   jurisdictional claims in published maps and institutional affiliations.

Supplementary Material

   Supplementary Table 1
   [174]srep44166-s1.doc^ (37.5KB, doc)

Acknowledgments