An Insight into the Behaviour of Recalcitrant Seeds by Understanding Their Molecular Changes upon Desiccation and Low Temperature

Round 1

Reviewer 1 Report

The abstract gives good context and summarises major conclusions. The introduction explains the problem well with references to the relevant literature. The structure here could be improved by moving or merging some paragraphs to reduce repetition. Later sections are also repetitive of some of the introduction's content. Changes between sections can be abrupt but there is an overall logic to the structure of the review. Perhaps conclude the introduction section by explaining the structure of the rest of the review to the reader. I am not convinced by the use of the term "stress" in later sections as desiccation and cold temperatures are anticipated by orthodox seeds and used as signals for normal development of dormancy. Later, the overall organisation into sections remains good but organisation within sections could be improved. For example, the transcriptomics section splits results for different species across the section and only defines gene functions near the end of the section. Tables and figures provide good summaries of key concepts and references. A concise conclusions sections sums up the main challenges in the field nicely to end the paper. Overall, this is a promising review that brings an updated perspective to the challenge of seed recalcitrance. Careful revision to reduce repetition and better organise material within sections would improve it further.  

Specific comments
L21-22 Was LEA specifically induced in recalcitrant species or is it a general response upon seed treatment?
L39 Give a time range for these "longer periods".
L54-74 These two pragraphs make repetitive statement about seed water content. Perhaps they could be combined and shortened.
L72 "-1" needs to be made uppercase.
L75 The switch in units between g/g-1 and % is confusing. Better to keep the same units throughout.
L99 Add a line explaining how knowledge of these papers can contribute to this study.
L107-115 Most of this paragraph is repetition from earlier. Perhaps reduce the earlier descriptions to keep the orthodox/recalcitrant comparison here.
L126 Figure 1 could be improved by giving range values to each of the characteristics listed.
L134 Typo: I do not know what you mean by "glass" here.
L154 Use present tense of "emerged"
L158 Clarify what the statement "This make these species inefficient large plantation for crop production" means.
L158-164 Repetition that should be reduced.
L165-167 Explain why cryopreservation works when ordinary freezing does not.
L196 Remove repeated "during seed desiccation"
L205 "Resolved" suggests that K+ efflux is protective of stress which is the opposite of what i think you mean to express.
L246-248 I do not see how knowledge of "G-C content, heterozygozity, and estimated genome size" could not add much to the study ofseed recalcitrance.
L257 Italicise "Panax"
L266 Typo: drop "that"
L276 What species does "Cs" refer to here if the initials of Chinese cork oak are Qv?
L279 Typo: change "response" to "respond"
L284-287 Move this explanation of LEA function to earlier in the section when LEA gene family members are first mentioned.
L288-296 This example describes TR-qPCR experiments for transciption but discusses hormone levels. Clarify what was tested and move this example to metabolomics section if necessary.
L308-309 and 313 Explain the acronyms MALDI-TOF MS, MS/MS LC at first use.
L361-362 What species does this study refer to?
L390 Typo: change "this" to "these"
L410 Typo: change "used" to "use"
L435-438 "species" is used repetitively in this statement.

 

Please see my specific comments in the suggestions for authors section.

Author Response

1. L21-22 Was LEA specifically induced in recalcitrant species or is it a general response upon seed treatment?

R1: Thank you so much for the question. Yes, this LEA was specifically induced in recalcitrant species through genomics and transcriptomics analyses which were highly expressed after exposure to desiccation and low temperature. However, LEA genes can also be found in orthodox species when these species encounter a developmentally regulated dehydration period.   

 

2. L39 Give a time range for these "longer periods".

R2: Thank you so much for the suggestion. Orthodox seeds with low water content, acquire desiccation tolerance during development and can be stored dry for longer periods of time, they can be kept under ambient conditions for 5-10 years half-life time. Meanwhile, in 40-60 years range under more optimal conditions [1,2].

 

3. L54-74 These two pragraphs make repetitive statement about seed water content. Perhaps they could be combined and shortened.

R3: Thank you for your suggestion. The crucial factors determining seed longevity are water content, temperature, and relative humidity [3]. Any changes that occur to the seed water content may impact the storability and longevity of seeds. [4]. Hence, maintaining seed viability is vital as the seed quality can significantly affect the uniformity of development (germination and dormancy), yield, and quality of the harvested crops. Furthermore, food security and crop production depend on maintaining the seed’s quality and viability after storage (in seed banks).

Excessive loss of water content or drought, exposure to extreme temperatures (high and ultra-low), and other abiotic stresses such as salinity, light, and heavy metal toxicity give a huge impact on seed viability [5]. These stresses that persist in plants may promote metabolic responses which will regulate their growth and development as well as for survival in harsh environmental conditions by producing a vast variety of flexible and adaptable regulators [6]. For example, the water content for storing of six different Brassicaceae species ranged from 0.02 to 0.03 g×H₂O×g^-1 [7]. Interestingly, for a variety of species, the critical water content of orthodox and recalcitrant seeds overlaps between 0.20 and 0.3 g H2O g^-1 DW [8].

 

4. L72 "-1" needs to be made uppercase.

R4: Thank you for the comment. The correction has been made in the paragraph from the number 3 section.

 

5. L75 The switch in units between g/g-¹ and % is confusing. Better to keep the same units throughout.

R5: Thank you for the suggestion. All water content values with g g-¹ unit have been converted to %.

 

6. L99 Add a line explaining how knowledge of these papers can contribute to this study.

R6: Thank you so much for your suggestion.  Some reviews on recalcitrant species utilizing omics analysis have previously been published by the author’s research group [9,10]. In this review, Table 1 shows there are several papers found in the Web of Science (WOS) and Google Scholar databases that are reporting on the use of omics approaches on several recalcitrant species. These reports may provide a comprehensive comprehension of the molecular mechanisms of recalcitrant species. This involves analyzing from all levels, ranging from genes to metabolites through omics analysis in order to gain a deeper understanding.

 

7. L107-115 Most of this paragraph is repetition from earlier. Perhaps reduce the earlier descriptions to keep the orthodox/recalcitrant comparison here.

R7: Thank you for the suggestion. The correction has been made in comment 3. so that we can keep the orthodox/recalcitrant comparison here.

 

8. L126 Figure 1 could be improved by giving range values to each of the characteristics listed.

R8: Thank you for your suggestion.

 

Figure 1. Comparison between orthodox and recalcitrant seeds that contribute to their distinct storage behaviours

 

9. L134 Typo: I do not know what you mean by "glass" here.

R: Apologies for the confusion. The non-reducing sugars and LEA proteins form intracellular glass, which then reduces the molecules' uptake across the cytoplasm and eventually limits chemical reactions [10]. Intracellular glass is also known as cytoplasmic glass or vitrified cytoplasm, is a phenomenon observed in certain plant seeds during desiccation. It refers to the transformation of the cytoplasm within the cells into a glassy or amorphous state when the seed is subjected to extreme dehydration [11].

 

10. L154 Use present tense of "emerged"

R10: Thank you for the comment. There are a number of challenges and problems that emerge when dealing with recalcitrant seeds.

 

11. L158 Clarify what the statement "This make these species inefficient large plantation for crop production" means.

R11: This sentence has been paraphrased to avoid confusion. Furthermore, species with recalcitrant seeds usually have a slow growth rate and fruit yield.
As a consequence, the large-scale cultivation of this species for crop production would be inefficient [12].

 

12. L158-164 Repetition that should be reduced.

R12: Thank you for the comment. On top of that, seed viability can easily decrease when water content drops to a certain percentage [13]. Specifically, recalcitrant seeds can lose their viability when it declines to 24-35.5% [14]. Moreover, the absence of a maturation-drying phase as seeds are metabolically active during their development [15]. Many tropical crops, such as coconut (Cocos nucifera), durian (Durio zibethinus), and mangosteen (Garcinia mangostana) have recalcitrant seeds which make propagation and also conservation difficult.  

 

13. L165-167 Explain why cryopreservation works when ordinary freezing does not.

R13: Thank you for the suggestion. Cryopreservation is a more promising alternative for storing recalcitrant seeds because this method utilizes specific protocols and cryoprotectants to minimize ice crystal formation helps prevent cellular damage and preserves the integrity of biological samples [16].

 

14. L196 Remove repeated "during seed desiccation"

R14: Thank you so much for the comment. However, during seed desiccation, the metabolic balance is interrupted as the production and accumulation of ROS become out of control (Figure 2).

 

15. L205 "Resolved" suggests that K+ efflux is protective of stress which is the opposite of what i think you mean to express.

R15:Thank you for the comment. Principally, electrolyte leakage is related to potassium ion, K⁺ efflux from plant cells, which is regulated by plasma membrane cation conductivity.

 

16. L246-248 I do not see how knowledge of "G-C content, heterozygozity, and estimated genome size" could not add much to the study of seed recalcitrance.

R16: Thank you for your comment. There is a shortage of molecular genetics knowledge for this recalcitrant mangosteen plant, which hinders genetic studies and crop development for this commercially significant fruit tree. Therefore, findings from genomics studies can help to identify specific genes that are involved in the recalcitrant nature of certain species. By comparing gene expression patterns between recalcitrant and non-recalcitrant species, researchers can pinpoint genes that are differentially expressed and potentially associated with recalcitrance. This information can shed light on the molecular pathways and regulatory networks involved in recalcitrant behaviour.

 

17. L257 Italicise "Panax"

R17: Transcriptomic analysis on recalcitrant seeds of Panax notoginseng was studied by Yang et al. [17].

 

18. L266 Typo: drop "that"

R18: Another transcriptomics analysis on recalcitrant seed of tea conducted by Jin, et al. [18], revealed expression profiles of 12 selected genes, related to seed dehydration treatment of the recalcitrant species.

 

19. L276 What species does "Cs" refer to here if the initials of Chinese cork oak are Qv?

R19: Cs here stands for Camellia sinensis, a species of tea plant. Hence the genes name, CsLEA genes.

 

20. L279 Typo: change "response" to "respond"

R20: CsLEA genes respond to low temperatures, indicating their involvement in abiotic stress tolerance.

 

21. L284-287 Move this explanation of LEA function to earlier in the section when LEA gene family members are first mentioned.

R21: Thank you for the suggestion. The section is moved to where the LEA gene family is first mentioned.

 

22. L288-296 This example describes RT-qPCR experiments for transcription but discusses hormone levels. Clarify what was tested and move this example to metabolomics section if necessary.

R22: Thank you for the comment and suggestion. This example has been clarified as follows, “RNA sequencing with RT-qPCR on mangosteen seed germination from day 0 to day 7 was conducted to analyze differentially expressed genes (DEGs) which help to understand the molecular mechanism of this recalcitrant seed during germination [19]. This study has revealed that abscisic acid (ABA) signalling has a role in stress response whereas gibberellin (GA) promotes growth potential during mangosteen seed germination. It was found on day 3, during the germination of mangosteen seeds, an increase in active transcripts leads to an elevation of ABA in the upregulated DEGs of day 3. On the other hand, among all phytohormones, GA exhibited the highest percentage of active transcripts, experiencing a significant decline on day 3.”

 

 

23. L308-309 and 313 Explain the acronyms MALDI-TOF MS, MS/MS LC at first use.

R23: Proteomics analysis was conducted using 2-D gel electrophoresis, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) or tandem mass spectrometry (MS/MS) analysis, and non-redundant searching of the National Resource for Biotechnology Information (NCBI) putative protein database revealed 23 proteins that associated with defense response, metabolism, and redox status upon desiccation exposure [20].

 

24. L361-362 What species does this study refer to?

R24: This study discusses the important roles of secondary metabolites generally found in plants which include orthodox species, Glycine max and recalcitrant species, Camellia sinensis.

 

25. L390 Typo: change "this" to "these"

R25: These findings can be affirmative to reveal the recalcitrance behaviour of the Quercus robur.

 

26. L410 Typo: change "used" to "use"

R26: Other than that, Fukushima et al. [21] highlighted the use of genome-scale metabolic reconstruction using modelling and mathematical simulations.

 

27. L435-438 "species" is used repetitively in this statement.

R27: Systems biology has the potential to improve our understanding of biological changes in recalcitrance as this desiccation-sensitive and low temperature-sensitive characteristic has hindered the effort of conservation and storage of plant species with this recalcitrance behaviour.

Author Response File: Author Response.docx

Reviewer 2 Report

This is a very interesting and meaningful topic. The author has summarized the differences in dehydration tolerance in seeds, which is very significant. Can we try to answer the following two questions? Because I believe that these questions are also of interest to many readers: 1. Can the conclusion that the important characteristic of recalcitrant seeds is their larger size compared to tolerant seeds be further supported by additional statistical data? 2. During the dehydration process, many gene expressions in seeds are altered. Can the author list and summarize the key genes that have been identified so far to have an impact on these seed tolerance differences? If there are such genes, what are their regulatory mechanisms?

Author Response

1. Can the conclusion that the important characteristic of recalcitrant seeds is their larger size compared to tolerant seeds be further supported by additional statistical data?

 

R: Thank you for the suggestion. Additional information of the statistical data of the size of both recalcitrant and orthodox seeds are included in the paragraph. “Differences between long-lived (orthodox) or short-lived (non-orthodox) species can be identified based on their size and water content. Examples of crop plants that bear orthodox seeds are wheat (Triticum aestivum L.), cereal rye (Secale cereale L.), legumes such as common bean (Phaseolus vulgaris L.), rice (Oryza sativa), banana (Musa balbisiana), and guava (Psidium guineense) [22-25]. Apart from their capability to retain viability when dried to low water content of 2%-5%, orthodox seeds can be also distinguished from recalcitrant seeds based on their size which is relatively smaller than recalcitrant seeds, as shown in Figure 1 [26]. Rapeseed plants (Brassica napus oleifera L.) bear orthodox seeds which size are ranged from 1.5-2.0 mm [27]. In addition, paddy plants which also bear orthodox seeds have seed length ranging from 8.61-11.29 mm [28]. On the other hand, recalcitrant seeds such as from Garcinia species have seed length ranging from 1.1-2.5 cm [29]. On top of that, large recalcitrant coconut (Cocos nucifera L.) seeds length ranging from 19.3-27.8 cm [30].”

 

 

1. During the dehydration process, many gene expressions in seeds are altered. Can the author list and summarize the key genes that have been identified so far to have an impact on these seed tolerance differences? If there are such genes, what are their regulatory mechanisms?

 

R: Thank you for the comment and suggestion. Some of the identified genes during the seed dehydration process are listed and summarized in a table and paragraph as follow.

 

 

Table 2: Genes that are involved during seed desiccation and their examples.

 

Genes	Examples	References
LEA genes	CsLEA genes   LEA-1, LEA-2, LEA-3, LEA-4, LEA-5, LEA-6	[27]   [77]
Dehydrin genes	QrDhn1, QrDhn2, QrDhn3, DN949901, Qp_Dhn1, Qp_Dhn2, Qp_Dhn3, Qp_Dhn4, Qp_Dhn5, Qp_Dhn6, Qp_AM711636, Qp_AM711635	[79]
ABA-responsive genes	ABI3, ABI5, AREBI, AREB2, RD29A, RD29B	[81]
Antioxidant genes	SOD1, APX1, CAT1, PDH1	[83]
Storage protein genes	Zein genes, oleosin genes, legumin and vicilin genes	[85] [86] [87]
Aquaporin genes	TIP3;1, TIP3;2,  GmPIP2;9, OsPIP1;1,   ZmPIP1;, AtNIP4;1, AtNIP4;2,  CsSIP2;1 and CsXIP	[91] [90]

 

R: During the desiccation process, some gene expressions in seeds have been altered and various genes that play critical roles in regulating the response to water loss and the preservation of seed viability. Several gene families and pathways are known to be involved in desiccation tolerance in seeds including late embryogenesis abundant (LEA) genes that are specifically linked to desiccation tolerance and have a vital function in protecting cells and cellular constituents during the process of seed desiccation. Some examples of LEA genes involved in desiccation or drying are CsLEA genes, LEA-1, LEA-2, LEA-3, LEA-4, LEA-5, LEA-6  [31,32].  These genes are expressed during seed desiccation and believed to protect cellular structures from damage caused by water loss. Moreover, dehydrins (DHNs) the group 2 of LEA (late embryogenesis abundant) proteins are induced under water deficit [41]. They are constituent elements of the developmental process in orthodox seeds, but also have been identified in recalcitrant seeds such as Acer saccharinum, Aesculus hippocastanum, Araucaria angustifolia, Camellia sinensis, Castanea sativa, and Poncirus trifoliate [33]. Examples of dehydrin genes identified under water deficit are QrDhn1, QrDhn2, QrDhn3, DN949901, Qp_Dhn1, Qp_Dhn2, Qp_Dhn3, Qp_Dhn4, Qp_Dhn5, Qp_Dhn6, Qp_AM711636, and Qp_AM711635 [33].  

Along with LEA and dehydrin genes, ABA genes also play an important role in water deficit response. Stomatal closure in guard cells is aided by ABA controlling solute efflux and regulates the expression of numerous genes and some of which may play a role in enhancing tolerance to dehydration in both plant leaves and seeds [42]. According to Nakashima et al. dehydration-responsive elements (DREs) act as connecting elements between ABRE and the expression of RD29A when responding to ABA [34]. From the findings, it was found from an expression analysis using abi3 and abi5 mutants showed that ABI3 and ABI5 play important roles in the expression of RD29B in seeds. Furthermore, as seeds undergo desiccation, they can experience oxidative stress due to the accumulation of reactive oxygen species (ROS) [43]. Antioxidant genes, such as SOD1, APX1, CAT1, PDH, help mitigate oxidative damage caused by ROS and maintain seed viability during drying [35]. The functions of catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and proline might have synergistically interacted to scavenge ROS  [43].

There are genes that responsible for storage proteins play vital roles in seed desiccation, as they are involved in gathering and storing proteins that serve as source of nutrients for the developing embryo during germination [44]. These storage proteins are crucial for seed development and early seedling growth. One of the examples is zein genes in maize, a class of storage proteins found in maize (corn) seeds and play a vital role in seed development and germination [36]. In addition, their content of proline enhances the protein's stability and safeguards it from desiccation stress. Another example of genes that encode storage protein are oleosin genes in oilseeds such as rapeseed, sunflower seeds, and sesame seeds [37]. They are associated with lipid bodies and play a role in storing and safeguarding oil reserves during seed desiccation. Meanwhile, legumin and vicilin genes are genes that encode for two major classes of storage proteins found in legume seeds such as soybeans and beans [38]. They act as a storage of nitrogen and amino acids for the developing embryo during the germination of the seed [44].

Seed dormancy changes are predominantly induced by temperature as well as water stress [45]. During water stress, aquaporins, which are membrane transport proteins, regulate water movement across cell membranes [46]. Some specific aquaporins are expressed in seeds during desiccation and contribute to controlling water loss and rehydration processes [40]. Examples of aquaporin genes that are expressed in seeds during desiccation are TIP3;1, TIP3;2, GmPIP2;9, OsPIP1;1, ZmPIP1;, AtNIP4;1, AtNIP4;2, CsSIP2;1 and CsXIP [39,40]. During seed germination, certain TIPs, such as TIP1s, have been observed to play a role in vacuole biosynthesis, facilitating water movement into vacuoles [40]. This process leads to the mobilization of reserve substances, the maintenance of cell turgor pressure, and the promotion of embryo cell elongation. Moreover, several PIPs, including PIP1s and PIP2s, are involved in water exchange between extracellular and cytoplasmic compartments, being essential for maintaining water balance within the cytoplasm.

Author Response File: Author Response.docx

Reviewer 3 Report

This review provides a detailed discussion on elucidating molecular responses and metabolic changes of recalcitrant seeds species and contains a wealth of useful information. However, some improvements are still needed.

In the section "1.2. Mechanism of Metabolic Changes in Seeds upon Stress", the author's description of "Reserves Accumulation" is basically limited to some carbohydrate substances, the content is not comprehensive enough, changes in protein and oil should also be summarized. In addition, this section only mentions the changes in reserves accumulation and ROS production and electrolyte flux, an additional overview on the changes in secondary metabolites, or other small molecule metabolites is needed.

 

Author Response

R:         Reserves or storage compounds such as carbohydrates, proteins, and oils can be accumulated by seeds abundantly which then serves as the main source of nutrients to initiate seed germination. Apart from being reserves, the production of these compounds act as plant’s mechanism in respond to biotic and abiotic stresses. Plants synthesize various classes of sugars and sugar alcohols as a response to desiccation [47]. For example, sucrose, raffinose, stachyose and cyclitols were accumulated in seed tissues of Inga vera, Caesalpinia echinata and Erythrina speciosa after subjected to different levels of drying [48]. Interestingly, those compounds were discovered to have higher concentrations in desiccation-tolerant C. echinate and E. speciosa than in recalcitrant I. vera [48]. Sugars and sugar alcohols were found not only act as osmoprotectants, but also as antioxidants in response to oxidative stress that caused by environmental changes [49]. Trehalose was observed to accumulate in the embryonic axis of E. speciosa, suggesting that it may have a significant impact on desiccation tolerance [50]. The compound accumulations indicate substantial relationship between seeds' storage behaviour and sugar metabolism and help to increase seed resistance to drying and freezing [51,52]. Additionally, trehalose is considered to be involved in the vitrification of seeds, which slows down enzymatic activities and helps to protect seeds' membranes from damage and cellular component deterioration [53].

 

            In addition to carbohydrates accumulation as storage compounds, there is oil content and lipids to serve as reserves in seeds. Studies conducted on recalcitrant seed, soursop (Annona muricata) seed oil found that the the average polyunsaturated fatty acids (PUFA) content varied from 31.72% in sundried seeds to 30.92% after 30 hours of oven-drying, and the drying process did not have a significant impact on it [54]. On the other hand, slow drying conducted on the intermediate seed, coffee seed (Coffea arabica) showed a significant increase of the concentration of free fatty acids (FFA) content [55]. These findings explain that the concentration of accumulation of oil content and lipids as reserves in seeds may vary based on the seed category and the type of treatment conducted.  Furthermore, drying and high temperature have significant effect on the changes of moisture content in seed which then would negatively affect the seed viability [56]. This may be supported by  biochemical analyses of seed proteins isolated that were conducted on soybean (Glycine max) seeds which priorly regulated under 22 ◦C  (control), 24 ◦C (moderate), and 26 ◦C (extreme) day/night temperatures [57]. It was reported that the accumulation of lipoxygenase, the β-subunit of β-conglycinin, sucrose binding protein and Bowman-Birk protease inhibitor have been deteriorated by the extreme heat stress.  

 

            Low temperature can also affect the composition of other compounds or metabolites along with carbohydrates, proteins, and fatty acids in the seeds. Findings from Xue et al. [58] revealed that, between the different incubation temperature, 15 ◦C, 25 ◦C and 30 ◦C on the germination of pecan seeds, (Carya illinoensis), incubated seeds under 30 ◦C is at which the metabolites showed the most significant differences. Hub metabolites found were mostly related to amino acids which include, valine, threonine, serine, lysine, citrulline, 3-hydroxy- proline, phenylalanine, methionine and ornithine. Apart from amino acids, organic acids such as oleic acid, linoleic acid, malonic acid, and palmitic acid were also significantly found in seeds that were incubated under 30 ◦C.  

Author Response File: Author Response.docx