Lysine 98 in NAC20/NAC26 transcription factors: a key regulator of starch and protein synthesis in rice endosperm.

Starch and proteins are main storage product to determine the appearance, cooking, texture, and nutritional quality of rice (Oryza sativa L.). OsNAC20 and OsNAC26, as pivotal transcription factors, redundantly regulate the expression of genes responsible for starch and protein synthesis in the rice endosperm. Any knockout of OsNAC20 or OsNAC26 did not result in visible endosperm defects. In this study, we had isolated and characterized a mutant named as floury endosperm25 (flo25). The caryopsis of the flo25 mutant exhibits a floury endosperm, accompanied by reductions in both the 1000-grain weight and grain length, as well as diminished levels of total starch and protein. Through map-based cloning, it was determined that FLO25 encodes a NAM, ATAF, and CUC (NAC) transcription factors, namely OsNAC26, with a lysine to asparagine substitution at position 98 in the flo25 mutant. Remarkably, lysine 98 is conserved across plants species, and this mutation does not alter the subcellular localization of OsNAC26 but significantly attenuates its transcriptional activity and its ability to activate downstream target genes. Furthermore, the mutant protein encoded by OsNAC26-flo25 could interact with OsNAC20, disrupting the native interaction between OsNAC20 proteins. Additionally, when lysine 98 is substituted with asparagine in OsNAC20, the resulting mutant protein, OsNAC20(K98N), similarly disrupts the interaction between OsNAC26 proteins. Collectively, these findings underscore the pivotal role of Lysine 98 (K) in modulating the transcriptional activity of NAC20/NAC26 within the rice endosperm.

Understanding the amylose biosynthesis and regulation mechanisms in Tartary buckwheat by the endosperm transcriptome.

Starch serves as a crucial energy source for both plants and humans, predominantly synthesized and stored in endosperms, tubers, rhizomes, and cotyledons. Given the significant role of amylose in determining the quality of starchy crops, optimizing its content has become a key objective in current crop breeding efforts. Tartary buckwheat, a dicotyledonous plant, notably accumulates high levels of amylose in its endosperm, surpassing common cereals like rice and maize. However, the mechanisms underlying amylose accumulation, distribution, and regulation in Tartary buckwheat remain unclear. Here, amylose content was determined across various tissues and organs of Tartary buckwheat, identifying with the endosperm as the primary site for its biosynthesis and accumulation. RNA sequencing analysis of endosperms from different developmental stages identified 35 genes potentially involved in starch biosynthesis, with 13 genes showing high endosperm-specific expression, suggesting crucial roles in starch biosynthesis. Additionally, the transcription factor FtNF-YB2, which was specifically highly expressed in the endosperm, was discovered to enhance amylose synthesis. Moreover, promoters with potential endosperm-specific activity were identified, advancing our understanding of amylose regulation. Additionally, this study also demonstrates that brassinosteroids (BR) positively influence amylose biosynthesis in Tartary buckwheat endosperm. These findings provide essential insights into the mechanisms of understanding amylose biosynthesis, accumulation and regulation in Tartary buckwheat, offering significant implications for future breeding strategies.

Immunolocalization of hordein synthesis and transport in developing barley endosperm.

The spatial accumulation of hordeins in the developing endosperm of barley grains was examined by immunofluorescence microscopy (immunolight microscopy [iLM]) and immunoelectron microscopy (iEM) to establish the timing and subcellular pattern of hordein synthesis and deposition. The pattern seen for hordeins was compared to other abundant grain proteins, such as serpin Z4 and lipid transfer protein 1 (LTP1). Hordein accumulates throughout grain development, from 6 to 37 days post-anthesis (DPA). In contrast, serpin Z4 was present at 6 DPA, but the greatest synthesis and accumulation occurred during the middle of seed development, from 15 to 30 DPA. LTP1 accumulated later in seed development, from 15 to 30 DPA. Hordeins accumulated within the lumen of the endoplasmic reticulum (ER), were exocytosed from the ER membrane, and accumulated in protein bodies, which then fused either with the protein storage vacuoles or with other protein bodies, which also later fused with the protein storage vacuoles. iEM showed hordein, and LTP1 appeared not to traverse the Golgi apparatus (GA). Hordein, LTP1, and serpin Z4 colocalized to the same protein bodies and were co-transported to the protein storage vacuole in the same protein bodies. It is likely that this represents a general transport mechanism common to storage proteins in developing grains.

Proteomic Profiling of Cocos nucifera L. Zygotic Embryos during Maturation of Dwarf and Tall Cultivars: The Dynamics of Carbohydrate and Fatty Acid Metabolism.

There is a limited number of studies analyzing the molecular and biochemical processes regulating the metabolism of the maturation of Cocos nucifera L. zygotic embryos. Our research focused on the regulation of carbohydrate and lipid metabolic pathways occurring at three developmental stages of embryos from the Mexican Pacific tall (MPT) and the Yucatan green dwarf (YGD) cultivars. We used the TMT-synchronous precursor selection (SPS)-MS3 strategy to analyze the dynamics of proteomes from both embryos; 1044 and 540 proteins were determined for the MPT and YGD, respectively. A comparison of the differentially accumulated proteins (DAPs) revealed that the biological processes (BP) enriched in the MPT embryo included the glyoxylate and dicarboxylate metabolism along with fatty acid degradation, while in YGD, the nitrogen metabolism and pentose phosphate pathway were the most enriched BPs. Findings suggest that the MPT embryos use fatty acids to sustain a higher glycolytic/gluconeogenic metabolism than the YGD embryos. Moreover, the YGD proteome was enriched with proteins associated with biotic or abiotic stresses, e.g., peroxidase and catalase. The goal of this study was to highlight the differences in the regulation of carbohydrate and lipid metabolic pathways during the maturation of coconut YGD and MPT zygotic embryos.

Preferentially expressed endosperm genes reveal unique activities in wheat endosperm during grain filling.

BACKGROUND: Bread wheat (Triticum aestivum L.) endosperm contains starch and proteins, which determine the final yield, quality, and nutritional value of wheat grain. The preferentially expressed endosperm genes can precisely provide targets in the endosperm for improving wheat grain quality and nutrition using modern bioengineering technologies. However, the genes specifically expressed in developing endosperms remain largely unknown. RESULTS: In this study, 315 preferentially expressed endosperm genes (PEEGs) in the spring wheat landrace, Chinese Spring, were screened using data obtained from an open bioinformatics database, which reveals a unique grain reserve deposition process and special signal transduction in a developing wheat endosperm. Furthermore, transcription and accumulation of storage proteins in the wheat cultivar, XC26 were evaluated. The results revealed that 315 PEEG plays a critical role in storage protein fragment deposition and is a potential candidate for modifying grain quality and nutrition. CONCLUSION: These results provide new insights into endosperm development and candidate genes and promoters for improving wheat grain quality through genetic engineering and plant breeding techniques.

SUBSTANDARD STARCH GRAIN7 regulates starch grain size and endosperm development in rice.

Starch is synthesized as insoluble, semicrystalline particles within plant chloroplast and amyloplast, which are referred to as starch grains (SGs). The size and morphology of SGs in the cereal endosperm are diverse and species-specific, representing a key determinant of the suitability of starch for industrial applications. However, the molecular mechanisms modulating SG size in cereal endosperm remain elusive. Here, we functionally characterized the rice (Oryza sativa) mutant substandard starch grain7 (ssg7), which exhibits enlarged SGs and defective endosperm development. SSG7 encodes a plant-specific DUF1001 domain-containing protein homologous to Arabidopsis (Arabidopsis thaliana) CRUMPLED LEAF (AtCRL). SSG7 localizes to the amyloplast membrane in developing endosperm. Several lines of evidence suggest that SSG7 functions together with SSG4 and SSG6, known as two regulators essential for SG development, to control SG size, by interacting with translocon-associated components, which unveils a molecular link between SG development and protein import. Genetically, SSG7 acts synergistically with SSG4 and appears to be functional redundancy with SSG6 in modulating SG size and endosperm development. Collectively, our findings uncover a multimeric functional protein complex involved in SG development in rice. SSG7 represents a promising target gene for the biotechnological modification of SG size, particularly for breeding programs aimed at improving starch quality.

WRKY10 Regulates Seed Size through the miR397a-LAC2 Module in Arabidopsis thaliana.

In angiosperms, seed size is a critical trait that is influenced by the complex interplay between the endosperm and seed coat. The HAIKU (IKU) pathway, involving the transcription factor WRKY10, plays a crucial role in regulating seed size in Arabidopsis thaliana. However, the downstream targets of WRKY10 and their roles in seed size determination remain largely unexplored. Here, we identified LACCASE2 (LAC2), a laccase gene involved in lignin biosynthesis, as a new downstream target of WRKY10. We observed that the expression of LAC2 was upregulated in the mini3 mutant, which is defective in WRKY10. We demonstrated that WRKY10 directly binds to the promoter of miR397a, activating its expression. miR397a, in turn, represses the expression of LAC2. Genetic analyses revealed that a mutation in LAC2 or overexpression of miR397a partially rescued the small seed phenotype of the MINISEED3 (MINI3) mutant mini3. Conversely, the overexpression of LAC2 in the wild type led to a decrease in seed size. These findings suggest that LAC2 functions as a negative regulator of seed size, and its expression is modulated by WRKY10 through miR397a. Our study uncovers a novel WRKY10-miR397a-LAC2 pathway that regulates seed size in Arabidopsis, providing new insights into the complex regulatory network governing seed development in plants.

Environment found to explain the largest variance in physical and compositional traits in malting barley grain.

BACKGROUND: Starch is the most abundant constituent (dry weight) in the barley endosperm, followed by protein. Variability of compositional and potentially related physical traits due to genotype and environment can have important implications for the malting and brewing industry. This was the first study to assess the effects of genotype, environment, and their interaction (G × E) on endosperm texture, protein content, and starch traits corresponding to granule size, gelatinization, content, and composition, using a multi-environment variety trial in California, USA. RESULTS: Overall, environment explained the largest variance for all traits (ranging from 23.2% to 76.5%), except the endosperm texture traits wherein the G × E term explained the largest variance (45.0-86.5%). Our unique method to quantify the proportion of fine and coarse milled barley particles using laser diffraction showed a binomial distribution of endosperm texture. The number of small starch granules varied significantly (P-value < 0.05) across genotypes and environments. We observed negative correlations between total protein content and each of enthalpy (-0.70), total starch content (-0.54), and difference between offset and onset gelatinization temperature (-0.52). Furthermore, amylose to amylopectin ratio was positively correlated to volume of small starch granules (0.36). CONCLUSION: Our findings indicate that environment played a larger role in influencing the majority of starch-related physical and compositional traits. In contrast, variance in endosperm texture was largely explained by G × E. Maltsters would benefit from accounting for environmental contributions in addition to solely genotype when making sourcing decisions, especially with regards to total protein, total starch, enthalpy, and difference between offset and onset gelatinization temperature. © 2024 Society of Chemical Industry.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Passiflora spp. (Passifloraceae).

In this chapter, we report advances in tissue culture applied to Passiflora. We present reproducible protocols for somatic embryogenesis, endosperm-derived triploid production, and genetic transformation for such species knowledge generated by our research team and collaborators in the last 20 years. Our research group has pioneered the work on passion fruit somatic embryogenesis, and we directed efforts to characterize several aspects of this morphogenic pathway. Furthermore, we expanded the possibilities of understanding the molecular mechanism related to developmental phase transitions of Passiflora edulis Sims. and P. cincinnata Mast., and a transformation protocol is presented for the overexpression of microRNA156.

The biosynthesis of storage reserves and auxin is coordinated by a hierarchical regulatory network in maize endosperm.

Grain filling in maize (Zea mays) is intricately linked to cell development, involving the regulation of genes responsible for the biosynthesis of storage reserves (starch, proteins, and lipids) and phytohormones. However, the regulatory network coordinating these biological functions remains unclear. In this study, we identified 1744 high-confidence target genes co-regulated by the transcription factors (TFs) ZmNAC128 and ZmNAC130 (ZmNAC128/130) through chromatin immunoprecipitation sequencing coupled with RNA-seq analysis in the zmnac128/130 loss-of-function mutants. We further constructed a hierarchical regulatory network using DNA affinity purification sequencing analysis of downstream TFs regulated by ZmNAC128/130. In addition to target genes involved in the biosynthesis of starch and zeins, we discovered novel target genes of ZmNAC128/130 involved in the biosynthesis of lipids and indole-3-acetic acid (IAA). Consistently, the number of oil bodies, as well as the contents of triacylglycerol, and IAA were significantly reduced in zmnac128/130. The hierarchical regulatory network centered by ZmNAC128/130 revealed a significant overlap between the direct target genes of ZmNAC128/130 and their downstream TFs, particularly in regulating the biosynthesis of storage reserves and IAA. Our results indicated that the biosynthesis of storage reserves and IAA is coordinated by a multi-TFs hierarchical regulatory network in maize endosperm.

An epiQTL underlying asexual seed formation in Arabidopsis.

KEY MESSAGE: The DNA methylation status at an epigenetic quantitative trait locus in the Arabidopsis chromosome 2 is linked to the formation of apomictic-like endosperms. Seed development in most angiosperms is coupled to fertilization of the maternal gametes by two sperm cells. However, apomictic species can reproduce asexually via seeds. This trait is of great agricultural interest, as it would fix complex genotypes and allow for pollen-independent seed production. However, engineering full apomixis requires three independent processes: apomeiosis, parthenogenesis and autonomous endosperm development. While the first two have been successfully engineered in some crops, the formation of autonomous endosperms remains a challenge. Although it is known that this trait is under epigenetic control, such as of DNA methylation, the underlying mechanisms remain mostly undiscovered. Here, using epigenetic recombinant inbred lines, we identified an epigenetic quantitative trait locus in the Arabidopsis chromosome 2, which correlates with permissiveness for the formation of asexual seeds: hypomethylation at this genomic region allows the formation of larger autonomous endosperms. Importantly, the methylation at this locus only correlates with asexual seed size, and not to the size of sexual seeds or that of other organs. With this, we aim to show that screening for epialleles is a promising strategy to uncover loci underlying relevant traits and could pave the way to identifying genes necessary for the engineering of apomixis.

Deciphering the Transcriptional Regulatory Network Governing Starch and Storage Protein Biosynthesis in Wheat for Breeding Improvement.

Starch and seed storage protein (SSP) composition profoundly impact wheat grain yield and quality. To unveil regulatory mechanisms governing their biosynthesis, transcriptome, and epigenome profiling is conducted across key endosperm developmental stages, revealing that chromatin accessibility, H3K27ac, and H3K27me3 collectively regulate SSP and starch genes with varying impact. Population transcriptome and phenotype analyses highlight accessible promoter regions' crucial role as a genetic variation resource, influencing grain yield and quality in a core collection of wheat accessions. Integration of time-serial RNA-seq and ATAC-seq enables the construction of a hierarchical transcriptional regulatory network governing starch and SSP biosynthesis, identifying 42 high-confidence novel candidates. These candidates exhibit overlap with genetic regions associated with grain size and quality traits, and their functional significance is validated through expression-phenotype association analysis among wheat accessions and loss-of-function mutants. Functional analysis of wheat abscisic acid insensitive 3-A1 (TaABI3-A1) with genome editing knock-out lines demonstrates its role in promoting SSP accumulation while repressing starch biosynthesis through transcriptional regulation. Excellent TaABI3-A1Hap1 with enhanced grain weight is selected during the breeding process in China, linked to altered expression levels. This study unveils key regulators, advancing understanding of SSP and starch biosynthesis regulation and contributing to breeding enhancement.

New Frontiers in Potato Breeding: Tinkering with Reproductive Genes and Apomixis.

Potato is the most important non-cereal crop worldwide, and, yet, genetic gains in potato have been traditionally delayed by the crop's biology, mostly the genetic heterozygosity of autotetraploid cultivars and the intricacies of the reproductive system. Novel site-directed genetic modification techniques provide opportunities for designing climate-smart cultivars, but they also pose new possibilities (and challenges) for breeding potato. As potato species show a remarkable reproductive diversity, and their ovules have a propensity to develop apomixis-like phenotypes, tinkering with reproductive genes in potato is opening new frontiers in potato breeding. Developing diploid varieties instead of tetraploid ones has been proposed as an alternative way to fill the gap in genetic gain, that is being achieved by using gene-edited self-compatible genotypes and inbred lines to exploit hybrid seed technology. In a similar way, modulating the formation of unreduced gametes and synthesizing apomixis in diploid or tetraploid potatoes may help to reinforce the transition to a diploid hybrid crop or enhance introgression schemes and fix highly heterozygous genotypes in tetraploid varieties. In any case, the induction of apomixis-like phenotypes will shorten the time and costs of developing new varieties by allowing the multi-generational propagation through true seeds. In this review, we summarize the current knowledge on potato reproductive phenotypes and underlying genes, discuss the advantages and disadvantages of using potato's natural variability to modulate reproductive steps during seed formation, and consider strategies to synthesize apomixis. However, before we can fully modulate the reproductive phenotypes, we need to understand the genetic basis of such diversity. Finally, we visualize an active, central role for genebanks in this endeavor by phenotyping properly genotyped genebank accessions and new introductions to provide scientists and breeders with reliable data and resources for developing innovations to exploit market opportunities.

Intersectional Hybrids between Darrow's Blueberry (V. darrowii Camp) and Lingonberry (V. vitis-idaea L.).

An initial cross of V. darrowii 'Johnblue' (Darrow's blueberry) × V. vitis-idaea 'Red Sunset' (lingonberry) produced more than 30 true intersectional diploid hybrids as confirmed by molecular markers. The most vigorous of these hybrids was extensively evaluated. This hybrid, US 2535-A, was floriferous and morphologically intermediate to the respective parents. Examination of pollen suggested low male fertility. Numerous crosses using the hybrid as a female reflected similarly low fertility and potential crossing barriers. Stylar examination suggested blockage of pollen tube growth in self-pollinations and significantly retarded growth in backcross pollinations. Nonetheless, two confirmed hybrid offspring were produced using the F1 hybrid as a female in crosses with V. vitis-idaea and V. darrowii, respectively. In a second set of crosses utilizing additional V. darrowii and V. vitis-idaea genotypes, another 23 verified hybrids in seven parental combinations were produced. Hybrids such as the ones presented offer the potential for generating de novo interspecific fruit types in blueberry and/or broadening the adaptation of lingonberry.

The seed morphospace, a new contribution towards the multidimensional study of angiosperm sexual reproductive biology.

BACKGROUND: The evolutionary success of flowering plants is associated with the vast diversity of their reproductive structures. Despite recent progress in understanding angiosperm-wide trends of floral structure and evolution, a synthetic view of the diversity in seed form and function across angiosperms is lacking. SCOPE: Here we present a roadmap to synthesise the diversity of seed forms in extant angiosperms, relying on the morphospace concept, i.e. a mathematical representation which relates multiple traits and describes the realised morphologies. We provide recommendations on how to broaden the range of measurable traits beyond mass, by using key morphological traits representative of the embryo, endosperm, and seed coat but also fruit attributes (e.g., dehiscence, fleshiness). These key traits were used to construct and analyse a morphospace to detect evolutionary trends and gain insight into how morphological traits relate to seed functions. Finally, we outline challenges and future research directions, combining the morphospace with macroevolutionary comparative methods to underline the drivers that gave rise to the diversity of observed seed forms. CONCLUSIONS: We conclude that this multidimensional approach has the potential, although still untapped, to improve our understanding of covariation among reproductive traits, and further elucidate angiosperm reproductive biology as a whole.

Two MADS-box proteins, AGL9 and AGL15, recruit the FIS-PRC2 complex to trigger the phase transition from endosperm proliferation to embryo development in Arabidopsis.

Spatiotemporal regulation of gene expression by polycomb repressive complex 2 (PRC2) is critical for animal and plant development. The Arabidopsis fertilization independent seed (FIS)-PRC2 complex functions specifically during plant reproduction from gametogenesis to seed development. After a double fertilization event, triploid endosperm proliferates early, followed by the growth of a diploid embryo, which replaces the endosperm in Arabidopsis and many dicots. Key genes critical for endosperm proliferation such as IKU2 and MINI3 are activated after fertilization. Here we report that two MADS-box AGAMOUS-LIKE (AGL) proteins associate with the key endosperm proliferation loci and recruit the FIS-PRC2 repressive complex at 4-5 days after pollination (DAP). Interestingly, AGL9 and AGL15 only accumulate toward the end of endosperm proliferation at 4-5 DAP and promote the deposition of H3K27me3 marks at key endosperm proliferation loci. Disruption of AGL9 and AGL15 or overexpression of AGL9 or AGL15 significantly influence endosperm proliferation and cellularization. Genome-wide analysis with cleavage Under Targets and tagmentation (CUT&Tag) sequencing and RNA sequencing revealed the landscape of endosperm H3K27me3 marks and gene expression profiles in Col-0 and agl9 agl15. CUT&Tag qPCR also demonstrated the occupancy of the two MADS-box proteins and FIS-PRC2 on a few representative target loci. Our studies suggest that MADS-box proteins could potentially recruit PRC2 to regulate many other developmental processes in plants or even in fungi and animals.

Insights into dynamic coenocytic endosperm development: Unraveling molecular, cellular, and growth complexity.

The endosperm, a product of double fertilization, is one of the keys to the evolution and success of angiosperms in conquering the land. While there are differences in endosperm development among flowering plants, the most common form is coenocytic growth, where the endosperm initially undergoes nuclear division without cytokinesis and eventually becomes cellularized. This complex process requires interplay among networks of transcription factors such as MADS-box, auxin response factors (ARFs), and phytohormones. The role of cytoskeletal elements in shaping the coenocytic endosperm and influencing seed growth also becomes evident. This review offers a recent understanding of the molecular and cellular dynamics in coenocytic endosperm development and their contributions to the final seed size.

Rice floury endosperm26 encoding a mitochondrial single-stranded DNA-binding protein is essential for RNA-splicing of mitochondrial genes and endosperm development.

Endosperm, the major storage organ in cereal grains, determines the grain yield and quality. Mitochondria provide the energy for dry matter accumulation, in the endosperm development. Although mitochondrial single-stranded DNA-binding proteins (mtSSBs) play a canonical role in the maintenance of single-stranded mitochondrial DNA, their molecular functions in RNA processing and endosperm development remain obscure. Here, we report a defective rice endosperm mutant, floury endosperm26 (flo26), which develops abnormal starch grains in the endosperm. Map-based cloning and complementation experiments showed that FLO26 allele encodes a mitochondrial single-stranded DNA-binding protein, named as mtSSB1.1. Loss of function of mtSSB1.1 affects the transcriptional level of many mitochondrially-encoded genes and RNA splicing of nad1, a core component of respiratory chain complex I in mitochondria. As a result, dysfunctional mature nad1 led to dramatically decreased complex I activity, thereby reducing ATP production. Our results reveal that mtSSB1.1 plays an important role in the maintenance of mitochondrial function and endosperm development by stabilizing the splicing of mitochondrial RNA in rice.

Transcriptional Control of Seed Life: New Insights into the Role of the NAC Family.

Transcription factors (TFs) regulate gene expression by binding to specific sequences on DNA through their DNA-binding domain (DBD), a universal process. This update conveys information about the diverse roles of TFs, focusing on the NACs (NAM-ATAF-CUC), in regulating target-gene expression and influencing various aspects of plant biology. NAC TFs appeared before the emergence of land plants. The NAC family constitutes a diverse group of plant-specific TFs found in mosses, conifers, monocots, and eudicots. This update discusses the evolutionary origins of plant NAC genes/proteins from green algae to their crucial roles in plant development and stress response across various plant species. From mosses and lycophytes to various angiosperms, the number of NAC proteins increases significantly, suggesting a gradual evolution from basal streptophytic green algae. NAC TFs play a critical role in enhancing abiotic stress tolerance, with their function conserved in angiosperms. Furthermore, the modular organization of NACs, their dimeric function, and their localization within cellular compartments contribute to their functional versatility and complexity. While most NAC TFs are nuclear-localized and active, a subset is found in other cellular compartments, indicating inactive forms until specific cues trigger their translocation to the nucleus. Additionally, it highlights their involvement in endoplasmic reticulum (ER) stress-induced programmed cell death (PCD) by activating the vacuolar processing enzyme (VPE) gene. Moreover, this update provides a comprehensive overview of the diverse roles of NAC TFs in plants, including their participation in ER stress responses, leaf senescence (LS), and growth and development. Notably, NACs exhibit correlations with various phytohormones (i.e., ABA, GAs, CK, IAA, JA, and SA), and several NAC genes are inducible by them, influencing a broad spectrum of biological processes. The study of the spatiotemporal expression patterns provides insights into when and where specific NAC genes are active, shedding light on their metabolic contributions. Likewise, this review emphasizes the significance of NAC TFs in transcriptional modules, seed reserve accumulation, and regulation of seed dormancy and germination. Overall, it effectively communicates the intricate and essential functions of NAC TFs in plant biology. Finally, from an evolutionary standpoint, a phylogenetic analysis suggests that it is highly probable that the WRKY family is evolutionarily older than the NAC family.