Abstract

   The transition from radicle emergence to seedling growth in maize is a
   crucial phase in the plant's life cycle, where rapid physiological and
   biochemical changes occur to facilitate successful development. In this
   study, we conducted a comparative transcriptomic analysis to gain a
   deeper understanding of the molecular processes driving this critical
   transition. The early divergence in gene expression patterns
   highlighted the upregulation of a substantial number of genes during
   radicle emergence. During radicle emergence, gene ontology (GO) term
   enrichment analysis unveiled active participation in biological
   processes such as chromatin assembly, cellular response to abiotic
   stress, and hormone signaling. This indicates that the initial stages
   of growth are marked by cellular expansion and adaptation to
   environmental stimuli. Conversely, in the seedling growth stage, GO
   analysis demonstrated a shift toward processes such as photosynthesis,
   nitrogen metabolism, and secondary metabolite biosynthesis, reflecting
   a transition to energy production and enhanced growth. In contrast,
   seedling growth was characterized by pathways related to photosynthesis
   and the production of gibberellins, crucial for robust seedling
   development. Hormonal regulation and starch metabolism were also
   prominent during radicle emergence, with various hormones, including
   auxins, diterpenoids, and brassinosteroids, driving processes like cell
   enlargement and stem growth. Moreover, starch and sucrose metabolism
   genes were expressed to mobilize stored reserves for energy during this
   stage. These findings offer valuable insights into the dynamic
   regulation of genes and pathways during this critical phase of maize
   development.

   Keywords: Maize, Radicle emergence stage, Seedling stage,
   Transcriptomics, Genes, Pathways

1. Introduction

   Zea mays L. is a prominent commercial and staple crop grown all over
   the world. The rate and uniformity of field crop establishment, which
   eventually determines the yield, particularly for maize, has a direct
   link to the early stages of plant growth i.e., seed germination and
   seedling establishment [[29]1]. The mature seed restarts growth
   throughout the intricate process of germination, changing its
   development plan from one driven by maturity to one that promotes
   seedling growth [[30]2]. A dry mature seed is expected to quickly
   absorb water (phase I), doing so until all of the seed tissues are
   moistened. Following phase II is a low intake of water, but phase III
   shows an increase in water uptake that coincides with the end stage of
   germination. The most important phase is Phase II, which is linked to
   many kinds of cellular and physiological functions, as well as DNA
   repair and the translation of both newly generated and stored mRNAs
   [[31][3], [32][4], [33][5]]. In fact, the final stage of seed
   germination is described as the extension of the embryonic axis, which
   often refers to radicles emerging out of the encasing seed layers
   [[34]6].

   The cell cycle must be activated for seedling development during the
   germination stage. Cell expansion in the embryonic axis is what largely
   drives seedling growth from the embryo. The majority of a plant's
   growth prospects after germination depend on cell divisions that take
   place in the adult plant embryo's root and shoot meristems. A crucial
   step in the development of a seedling is the proliferation of the
   embryo root meristem, which is required for the beginning of root
   growth [[35]7,[36]8]. It has been shown in the past that the start of
   the mitotic cell cycle occurs in the shoot and root meristems at the
   last stages of seed germination and is regulated by plant hormones.
   These modifications may be seen in the gene expression patterns, which
   abruptly show a transition from a germinative to a developmental
   program [[37]9].

   ISTA has certified the RE test (early single count of radicle
   emergence) to measure maize seed vigor [[38]10]. In many crop species,
   the RE test has been linked to seedling emergence potential
   [[39]11,[40]12]. The underlying physiology of the RE test is the time
   frame (lag period) between imbibition and radicle emergence, which
   reveals the restart of gene expression from the embryo's resting
   condition to establish a functioning genetic program allowing the new
   plant to develop. It includes signal transduction, cell wall remodeling
   and modification, phytohormone metabolism, a surge in metabolic
   pathways for energy availability, and the synthesis of macromolecules,
   mRNAs, and proteins that allow cells to survive and thrive [[41]13].
   The activation of the embryo root meristem is a critical phase of the
   post-germination stage, where radicles continue to elongate and
   cotyledons appear and expand. This is during which photosynthesis and
   energy metabolism processes are interconnected, leading to the
   establishment of the seedling [[42]14]. Seed tissues perform distinct
   activities during germination, which lasts around 66 h in maize
   [[43]15]. As a result, investigations of particular tissues at certain
   time points must be focused on to analyze the development process at
   the molecular level. In this situation, transcriptomics has shown to be
   a potent tool for examining certain biological functions of development
   in maize. Since the tissues that will grow and develop to make a new
   plant are present at both the initial radicle emergence stage and the
   final seedling stage in maize, an overall study of the expression at
   both steps has been carried out by RNA sequencing to set special
   emphasis on the various genes controlling several regulatory mechanisms
   during these crucial stages.

2. Materials and methods

2.1. Seed germinating condition and sample phenotyping

   In our research study, we employed seeds of the maize (Zea mays L.),
   specifically the hybrid MAH 14-5, as the biological system under
   investigation. These maize seeds were sourced from the AICRP on Seed
   (Crops), University of Agricultural Sciences, GKVK, Bengaluru. Notably,
   the seeds used in our experimentation were procured from a newly
   harvested, untreated seed lot, ensuring the freshness and integrity of
   the plant material. This deliberate choice of untreated seeds aimed to
   maintain the seed's natural state and characteristics, which is pivotal
   for unbiased scientific analysis.

   After being surface-sterilized in 70% (v/v) ethanol for 15 min, seeds
   were then three times rinsed in sterile distilled water [[44]16]. Seeds
   were kept for germination in a sterile glass plate covered with glass
   jars for 48–66 h by water imbibition on moisturized Whatman filter
   paper at 27 ± 1 °C in a growth chamber. Three biological replicates
   were prepared and ten seeds were mixed for each sample. The embryonic
   axes were excised manually from the radicle emergence stage; whereas in
   7-day-old seedlings root and shoot samples were collected separately
   for RNA isolation. The RNA was extracted as per the manufacturer's
   protocol using the Trizol reagent (Takara) as shown in [45]Fig. 1 (A).

Fig. 1.

   [46]Fig. 1
   [47]Open in a new tab

   (A) Pictorial representation of tissues selected for transcriptomic
   analysis during the radicle emergence stage and seedling stage, (B)
   Principal component analysis (PCA) plot of all expressed genes in the
   RNA−seq data. The X−axis indicates the first principal component; the
   Y−axis indicates the second principal component. The percentage of
   variance explained by each PC is shown in each case. 1–3, biological
   replicates, (C) Volcano plot of expressed genes of seedlings compared
   to radicle emergence stage. The y-axis illustrates −log10 p values and
   the x-axis corresponds to a log2-fold change of gene expression between
   seedlings and the radicle emergence stage. Significantly up and
   downregulated genes are highlighted in green and red colors,
   respectively. (For interpretation of the references to color in this