Abstract

   The kernel serves as a storage organ for various nutrients and
   determines the yield and quality of maize. Understanding the mechanisms
   regulating kernel development is important for maize production. In
   this study, a small-kernel mutant smk7a of maize was characterized.
   Cytological observation suggested that the development of the endosperm
   and embryo was arrested in smk7a in the early development stage.
   Biochemical tests revealed that the starch, zein protein, and
   indole-3-acetic acid (IAA) contents were significantly lower in smk7a
   compared with wild-type (WT). Consistent with the defective development
   phenotype, transcriptome analysis of the kernels 12 and 20 days after
   pollination (DAP) revealed that the starch, zein, and auxin
   biosynthesis-related genes were dramatically downregulated in smk7a.
   Genetic mapping indicated that the mutant was controlled by a recessive
   gene located on chromosome 2. Our results suggest that disrupted
   nutrition accumulation and auxin synthesis cause the defective
   endosperm and embryo development of smk7a.

   Keywords: maize, smk7a mutant, IAA, starch synthesis, transcriptome

1. Introduction

   Maize (Zea mays) is one of the most important crops in the world. The
   maize kernel is the storage organ that contains the essential
   components for plant growth and reproduction. The maize kernel consists
   of the embryo, endosperm, and surrounding pericarp. The embryo consists
   of the shoot apical meristem, root apical meristem, scutellum, and leaf
   primordium and has the potential to develop into a new plant [[40]1].
   The endosperm accounts for 85% of the kernel’s weight and stores about
   80% of the starch and 8–15% of the protein. The development of the
   endosperm begins with the fertilized central cells, followed by
   cellularization and differentiation into four main types of cells: the
   basal endosperm transfer layer (BETL), the aleurone layer (AL), the
   starchy endosperm (SE), and the embryo-surrounding region (ESR)
   [[41]2,[42]3,[43]4,[44]5,[45]6,[46]7]. Maize kernel development is a
   complex and dynamic process and is regulated by a large number of
   genes. With the development of molecular techniques, many genes that
   are crucial for kernel development have been identified, and the
   regulatory mechanisms have also been uncovered [[47]8].

   Regarding embryo development, a number of embryo-specific (emb) genes
   have been studied, such as LEM1, EMB8516, EMB14, EMB12, and EMB16
   [[48]9,[49]10,[50]11,[51]12,[52]13], all of which have been implicated
   in plastid function. These studies have demonstrated that plastid
   protein translation is essential to embryogenesis. Regarding endosperm
   development, the functional genes involved in the development of the
   major endosperm cell types have also been elucidated. The AL is a
   cuboidal cell layer that forms on the epidermal layer of maize
   endosperm cells. Several genes have been reported to control the fate
   of the AL cells via positional signaling, including CR4 [[53]14], DEK1
   [[54]15,[55]16], SAL1 [[56]17], and THK1 [[57]18,[58]19]. The BETL, a
   unique endosperm cell layer, plays an important role in nutrient
   transport and signaling between the filial and maternal tissues
   [[59]20]. There are two key genes that affect the nutrient uptake and
   partitioning in the BETL region. The first gene is MN1, which is
   located in the BETL and the pedicel (PED) and catalyzes the conversion
   of sucrose into glucose and fructose [[60]21,[61]22]. The second gene
   ZmSWEET4c encodes a sugar transporter [[62]23], and it can transport
   hexose to the starchy endosperm and embryo for its development
   [[63]24,[64]25]. ZmMRP-1 is a well-known transcriptional regulator in
   BETL differentiation. It controls the expression of BETL-specific
   genes, including ZmTCRR-1 and ZmTCRR-2 [[65]26,[66]27], MEG1 [[67]10],
   and BETL-1, BETL-2, BETL-9, and BETL-10. The SE is the major storage
   tissue for starch synthesis and protein storage in the endosperm.
   Starch biosynthesis requires multiple classes of enzymes, including
   adenosine diphosphate (ADP)-glucose pyrophosphorylase (AGPase), starch
   synthases (SSs), starch branching enzymes (SBEs), and starch
   debranching enzymes (DBEs) [[68]28]. The key genes have been identified
   in mutant studies, such as SH2, BT2, DU1, and SU1 [[69]29,[70]30]. The
   loss of the function of these genes results in disturbed starch
   synthesis and defective kernel development. The ESR is formed from
   small dense cytoplasmic cells surrounding the young embryo. The
   functions of the ESR are providing nutrition to the embryo, defense
   from pathogens, and signaling at the embryo-endosperm interface. The
   ESR1, ESR2, and ESR3 genes have been suggested to play an important
   role in regulating embryo–endosperm signaling [[71]31].

   Although significant progress has been made in studies on maize kernel
   development, additional knowledge is still needed to reveal the complex
   interaction network during embryo and endosperm development. In this
   study, a thorough analysis of a small kernel mutant smk7a was conducted
   using cytology, biochemistry, and transcriptomics. These results
   provide a foundation for further gene cloning and elucidation of the
   mechanism involved in maize kernel development.

2. Results

2.1. smk7a Mutant Exhibited Defective Embryo and Endosperm Development

   Compared with WT kernels, smk7a exhibited defective kernel development
   and a lower 100-kernel weight ([72]Figure 1A–C and [73]Figure S1). To
   observe the structural and cytological changes, we carried out paraffin
   sectioning on WT and smk7a kernels collected at 12, 16, 20, 25, and 30
   DAP. Longitudinal sections of the whole seed revealed that smk7a showed
   a small and shrunken kernel phenotype, with severely arrested
   embryogenesis and endosperm development. The WT embryo developed to the
   late embryogenesis stage, with established scutellum, coleoptiles, and
   shoot apical meristem (SAM) at 12 DAP; differentiated primary leaves at
   16 DAP; established root apical meristem (RAM) at 20 DAP; and good
   differentiation at 30 DAP. However, the development of the smk7a
   embryos was severely delayed at 12 DAP, and it did not reach the
   coleoptilar stage with differentiated scutellum, coleoptile, SAM, and
   RAM until 30 DAP ([74]Figure 1D). The WT endosperm was well developed
   and starch-filled from 12 to 30 DAP, while the endosperm of smk7a was
   small and underdeveloped, and a gap was observed between the endosperm
   and epidermis ([75]Figure 1D). Further observations revealed that the
   transfer cells in the BETL of the WT were large and exhibited clear
   cell wall ingrowths at 12 DAP, and the flange wall ingrowth became more
   dense at 20 DAP. In contrast, the BETL cells in the smk7a kernels were
   mostly small and lacked extensive cell wall ingrowth ([76]Figure 1E).

Figure 1.

   [77]Figure 1
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   Phenotype features of the maize smk7a mutant. (A) An F[2] ear of
   smk7a/WT at 12 DAP. The arrows indicate three randomly selected mutant
   kernels. The scale bar is 1 cm. (B) A mature F[2] ear of smk7a/WT. The
   arrows indicate three randomly selected mutant kernels. The scale bar
   is 1 cm. (C) Examples of mature WT and smk7a kernels from the
   segregated F[2]. The scale bar is 1 mm. (D) Light microscopy analysis
   of immature kernels from the WT and smk7a collected at 12, 16, 20, 25,
   and 30 DAP. The scale bar is 1 mm. En, endosperm; Em, embryo; SAM,
   shoot apical meristem; RAM, root apical meristem. (E) Light microscopy
   analysis of BETL cells of the WT and smk7a kernels collected at 12, 20,
   and 30 DAP. DAP, days after pollination. BETL, Basal endosperm transfer
   layer; PC, placenta. The scale bar is 200 μm.

   We also found that smk7a had a 56.3% reduction in the endosperm cell
   number compared to the WT, and the endosperm cell size was 188.7%
   larger than that of the WT at 20 DAP ([79]Figure 2A–C and [80]Table 1).
   Although the endosperm cell size was larger, the starch granules of
   smk7a appeared to be less filled than the endosperm of the WT
   ([81]Figure 2A). Scanning electron microscopy (SEM) was used to
   investigate the starch accumulation in the endosperm at 34 DAP. The
   results revealed that the endosperm cells of the WT were filled with
   starch granules, while there were few granules in the endosperm cells
   of smk7a, and irregular polygonal granules were observed ([82]Figure
   2E,F).

Figure 2.

   [83]Figure 2
   [84]Open in a new tab

   Comparison of the endosperm cells and starch granules in the WT and
   smk7a. (A) Light microscopy observation of the endosperm cells of the
   WT and smk7a kernels collected at 20 DAP. The scale bar is 100 μm.
   Comparison of the endosperm (B) cell area and (C) cell number between
   the WT and smk7a. The values are the means of the three replicates ±
   SD. Thirty cells were selected for statistical analysis for each
   replicate. ** denotes statistical significance at p < 0.01 according to
   the t-test. (D) Longitudinal section of the kernels collected from the
   WT and smk7a at 34 DAP. The red square indicates the region that was
   observed via scanning electron microscopy (SEM). The scale bar is 1 mm.
   (E) SEM analysis of the peripheral regions of the WT and smk7a
   endosperm collected at 34 DAP. The scale bar is 100 μm. (F) SEM
   analysis of the starch granules of the WT and smk7a endosperm. SG,
   starch granule. The scale bar is 10 μm.

Table 1.

   Comparison of the endosperm cell area and cell number between the WT
   and smk7a in the same field of vision.
                       WT      smk7a    Percentage Change    p Value
   Cell area (μm^2) 24,616.84 71,077.38      188.7%       9.06 × 10^−6
     Cell number       112       49          −56.3%       2.02 × 10^−3
   [85]Open in a new tab

2.2. smk7a Mutant Had Lower Starch, Storage Protein, and Free IAA Contents

   To further determine the underlying biochemical basis for the defective
   phenotype of the smk7a mutant, we measured the soluble sugar, total
   starch, and protein contents of the WT and smk7a kernels. Based on our
   results, the content of the soluble sugars (including glucose,
   fructose, and sucrose) of the smk7a kernels was 1.40–2.70-fold higher
   than that of the WT kernels at 12 DAP, and it was 3.72–4.07-fold higher
   at 20 DAP ([86]Figure 3A–C). In contrast, the starch content was
   significantly lower, i.e., 4.25-fold and 4.67-fold at 12 and 20 DAP,
   respectively ([87]Figure 3D). In addition, the starch content of smk7a
   was only 87.8% that of the WT in the mature stage ([88]Figure 3D). We
   also noticed that the total soluble sugar content of the WT decreased
   by 40.3% from 12 DAP to 20 DAP, while the starch content increased,
   which represents the balanced conversion of sugar to starch. However,
   for smk7a, when the starch content increased, the soluble sugar content
   also increased by 22.3% from 12 to 20 DAP. These results suggest that
   the sugar metabolism and starch synthesis were disturbed in the smk7a
   kernels in the filling stage. Quantitative analysis of the protein
   contents revealed that the zein protein was also significantly lower in
   smk7a and was 71.7%, 76.7%, and 94.0% that of the WT at 12 DAP, 20 DAP,
   and in the mature stage, respectively ([89]Figure 3E).

Figure 3.

   [90]Figure 3
   [91]Open in a new tab

   Comparison of biochemical values of kernels from the WT and smk7a
   mutant. (A–C) Comparison of the soluble sugar contents of the WT and
   smk7a at 12 and 20 DAP. Glc, glucose, Fru, fructose, Suc, sucrose. (D)
   Starch contents of the WT and smk7a at 12 and 20 DAP and in the mature
   stage. (E) Zein protein contents of the WT and smk7a at 12 and 20 DAP
   and in the mature stage. (F) Free IAA contents of the WT and smk7a at
   12 and 20 DAP. All of the values are the means of the three replicates
   ± SD. ** denotes statistical significance at p < 0.01, and * denotes
   statistical significance at p < 0.05, according to the t-test when
   compared with the values of the WT.

   IAA has been reported to play an important role in kernel development.
   We compared the free IAA contents of the WT and smk7a at 12 and 20 DAP.
   Based on our UHPLC-ESI-MS/MS analyses, the free IAA content of smk7a
   was 58.3% and 83.2% that of the WT at 12 DAP and 20 DAP, respectively
   ([92]Figure 3F). These results suggest that IAA synthesis was
   suppressed in smk7a in the early development stages.

2.3. Transcriptome Comparison of Developing Kernels from WT and smk7a

   To reveal the gene expression changes between the WT and the smk7a
   mutant, we conducted RNA sequencing (RNA-seq) using the kernels
   collected at 12 and 20 DAP. On average, 48.2 million clean reads were
   obtained for each sample, and 89.2–94.9% of the reads were uniquely
   mapped to the maize B73 reference genome ([93]Table S1). With a
   threshold fold change of >2 and an adjusted p value of <0.05, a total
   of 3405 and 2747 differentially expressed genes (DEGs) were identified
   between smk7a and WT for the 12 and 20 DAP kernels, respectively.
   Further classification of the DEGs revealed that there were 1522
   downregulated genes and 1883 upregulated genes at 12 DAP, and there
   were 1124 downregulated genes and 1623 upregulated genes at 20 DAP
   ([94]Tables S2–S3). Since the BETL development was impaired in smk7a,
   we checked the expression levels of the BETL-specific genes. At 12 DAP,
   six genes were significantly downregulated in smk7a, namely, BETL3,
   BETL4, BETL9, TCRR1, TCRR2, and SWEET4C, while only the SWEET4C gene
   was significantly downregulated at 20 DAP. The expression levels of
   BETL4, BETL9, TCRR1, and SWEET4C in smk7a and WT were further verified
   using the quantitative real-time polymerase chain reaction (qRT-PCR)
   method ([95]Figure S3A). The reduced expression of the BETL-specific
   genes in smk7a validated the development defects of the BETL cells.

   To associate functions with the DEGs, we performed gene ontology (GO)
   enrichment analysis. The 20 most significantly (p < 0.05) enriched GO
   terms in the biological process, molecular function, and cellular
   component categories are presented in [96]Figure S3. At 12 DAP, the
   downregulated genes were mainly enriched in the BP term “starch
   biosynthetic process”, the MF term “nutrient reservoir activity”, and
   the CC terms “chloroplast and amyloplast”, while the upregulated genes
   were mainly enriched in the BP terms “cell differentiation”, “lipid
   catabolic process”, and “hydrogen peroxide catabolic processes”, the MF
   term “heme binding”, and the CC term “extracellular region” ([97]Figure
   S2A). At 20 DAP, the downregulated genes were significantly enriched in
   the BP term “secondary metabolite biosynthetic process”, the MF term
   “nutrient reservoir activity”, and the CC term “extracellular region”,
   while the upregulated genes were mainly enriched in the BP terms
   “defense response” and “hydrogen peroxide catabolic processes”, the MF
   term “sequence-specific DNA binding”, and the CC terms “extracellular
   region” and “plant type cell wall” ([98]Figure S2B).

   We further conducted Kyoto Encyclopedia of Genes and Genomes (KEGG)
   enrichment analysis of the DEGs. The results revealed that the “starch
   and sucrose metabolism” and “plant hormone signal transduction” were
   the most significantly enriched pathways for the downregulated genes in
   both development stages ([99]Figure 4A,B), while the “fatty acid
   biosynthesis” and “glycolysis/gluconeogenesis pathways” were only
   enriched at 12 DAP ([100]Figure 4A). For the upregulated genes, the
   “phenylpropanoid biosynthesis” pathway was significantly enriched in
   the two development stages ([101]Figure 4A,B).

Figure 4.

   [102]Figure 4
   [103]Open in a new tab

   KEGG pathway enrichment analysis of differentially expressed genes
   between WT and smk7a at 12 and 20 DAP. (A) Top five KEGG enrichment
   pathways of the up- and down-regulated DEGs of smk7a compared to the WT
   at 12 DAP. (B) Top five KEGG enrichment pathways of up- and
   down-regulated DEGs of smk7a compared to the WT at 20 DAP. The Y-axis
   shows the KEGG pathway. The X-axis shows the −log 10 (q-value). The
   size of each dot represents the number of genes enriched in a
   particular pathway.

   These results suggest that the expression of the genes with starch
   synthesis and nutrient accumulation functions was suppressed in smk7a
   compared to that in the WT in both development stages. The suppressed
   transcriptional activity of these genes could be the major reason for
   the defective development of the smk7a endosperm and embryo.

2.4. Suppressed Expression of Genes Involved in Starch Synthesis and Protein
Storage in smk7a

   Starch and storage protein accumulation is essential for kernel
   development and yield formation. Our RNA-seq results revealed that the
   genes that participated in the “starch and sucrose metabolism” pathway
   were significantly downregulated in smk7a compared to that in the WT
   ([104]Figure 4). In the starch synthesis process, sucrose, glucose, and
   fructose are first imported into the endosperm cell and are metabolized
   to hexose phosphates. Then, these hexose phosphates serve as substrates
   for starch biosynthesis in the amyloplast [[105]32,[106]33]. The
   sucrose synthase SH1, SUS2, and SUS3 functions for converting sucrose
   into fructose and uridine-diphosphoglucose (UDPG) were downregulated in
   the smk7a by 6.9–13.9-fold at 12 DAP and 0.9–1.9-fold at 20 DAP. The
   hexokinase (HEX) phosphorylates hexoses forming hexose phosphate, HEX1,
   and HEX5 genes were downregulated in smk7a by 2.3–5.0-fold and
   1.0–1.8-fold at 12 and 20 DAP, respectively ([107]Figure 5A). The
   interconversions among fructose 6-phosphate (F6P), glucose 6-phosphate
   (G6P), glucose 1-phosphate (G1P), and UDPG were catalyzed by
   phosphohexose isomerase (PHI), phosphoglucomutase (PGM), and
   UDP-glucose pyrophosphorylase (UGP), and four related genes were also
   found to have a reduced expression in smk7a ([108]Figure 5A). The
   ADP-glucose pyrophosphorylase genes (APGs) catalyze the first step in
   starch biosynthesis, which is also the limiting step. Five APGs were
   significantly downregulated in smk7a, including SH2 and BT2, the
   expressions of which were decreased by 9.2- and 26-fold, respectively,
   at 12 DAP ([109]Figure 5A). During the starch synthesis in the
   amyloplast, the expressions of the genes with amylose synthase (such as
   WX1, GBSSI, and GBSSIb), amylopectin synthase (SSIV, SSV, and SSI),
   branching enzyme (SBE), and starch debranching enzyme (SU1) functions
   were all significantly reduced ([110]Figure 5A and [111]Table S4). To
   further verify these results, the expression levels of SH1, SH2, WX1,
   BT2, AGP, and SSI in smk7a and the WT were examined using the qRT-PCR
   method ([112]Figure S3B). The overall downregulation of the expression
   of the genes with starch and sucrose metabolism pathway functions was
   in agreement with the lower starch content in smk7a at 12 and 20 DAP
   and in the mature stage.

Figure 5.

   [113]Figure 5
   [114]Open in a new tab

   Heat maps of the DEGs that participate in the selected disrupted
   function or pathway. (A) Heat maps of DEGs enriched in starch and
   sucrose metabolism. The left side color bar represents 12 DAP, the
   right side color bar represents 20 DAP. (B) Heat maps of DEGs with the
   sugar transport function. (C) Heat maps of DEGs with IAA synthesis and
   signaling functions. The color bar indicates the log2 (fold change)
   values from small to large.

   We further checked the expression of the sugar transporter genes since
   the soluble sugar content was higher in smk7a compared to that in the
   WT. We detected 19 transporters, including SWEETs, sugar transporter
   proteins (STPs), and sucrose transporters (SUTs), which were expressed
   differently in smk7a and the WT, and 14 of these genes were upregulated
   in at least one development stage ([115]Figure 5B and [116]Table S5).
   These results suggest that suppressed starch synthesis may act as a
   feedback signal for stimulating sugar transport from the maternal
   tissue to the kernel endosperm, even when the development of the BETL
   cells is defective.

   Moreover, we also found that 23 genes related to protein storage under
   the GO term “nutrient reservoir activity” (GO: 0045735) were
   significantly downregulated in smk7a. It should be noted that central
   player transcript factors O2, O11, and PBF were also downregulated in
   smk7a in both development stages ([117]Figure 5C and [118]Table S6).
   These results provide a sufficient explanation for the reduced zein
   protein content in smk7a compared to that in the WT.

2.5. Disrupted Expression of Genes Related to IAA Synthesis and Signaling in
smk7a

   Studies have reported that phytohormones such as auxin, cytokinins, and
   brassinosteroids play an important role in plant seed development. In
   this study, the IAA content in smk7a was significantly lower, and our
   RNA-seq results also revealed that the expression of the key genes
   involved in auxin synthesis was decreased in smk7a ([119]Figure 6 and
   [120]Table S7). The tryptophan aminotransferase (TAA1/TAR) genes
   convert tryptophan to indole-3-pyruvic acid (IPA). Mutations in the
   TAA1/TAR of Arabidopsis lead to a dramatic reduction in the free IAA
   level. Three TAR genes were found to be downregulated in smk7a at 12
   DAP, while the expression level of TAR1 and TAR3 increased at 20 DAP.
   ZmYUC1, a flavin-monoxygenase, which is the key enzyme related to auxin
   biosynthesis in maize endosperm, was significantly downregulated (by
   6.1-fold) in smk7a at 12 DAP. The suppressed IAA synthesis also caused
   a disturbance of the IAA signaling. The expression levels of the core
   auxin signaling factors, including 10 auxin/indole-3-acetc acid
   (Aux/IAA), five auxin responsive factors (ARFs), six Gretchen Hagen3
   (GH3), and seven small auxin RNA (SAUR) were changed in smk7a
   ([121]Figure 6). In the presence of low-level intracellular auxin,
   AUX/IAA proteins inhibit the function of ARF proteins. Based on our
   RNA-seq, nine of the 10 AUX/IAAs were upregulated in smk7a, while all
   five ARFs were downregulated in smk7a ([122]Figure 6). These results
   suggest that auxin synthesis and signaling were suppressed in smk7a,
   which may be the major cause of the reduced number of cells in the
   smk7a endosperm.

Figure 6.

   [123]Figure 6
   [124]Open in a new tab

   Heat maps of DEGs enriched in IAA synthesis and signaling functions.
   The color bar indicates the log2 (fold change) values from small to
   large. The left-side color bar represents 12 DAP, the right-side color
   bar represents 20 DAP.

2.6. Genetic Analysis and Fine Mapping of the smk7a Mutant

   Our previous study reported that smk7a mutant was controlled by a
   recessive gene located in the 120 kb region on chromosome 2 [[125]34].
   To further narrow down its location, we genotype another F[2]
   population of 1056 homozygous mutant kernels using newly developed
   insertion and deletion markers ([126]Table S8); however, no new
   recombination was detected at this interval. The RNA-seq results
   revealed that only three genes exhibited an expression level greater
   than 1 fragment per kilobase of the transcript per million fragments
   (FPKM) in at least one of the samples in this region. Zm00001d001819
   encodes an N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase,
   Zm00001d001824 encodes a Dof zinc finger protein, and Zm00001d001825
   encodes a transducin/WD40 repeat-like superfamily protein.

3. Discussion

   The maize kernel has been used extensively as a valuable model for
   cereal kernel development studies. The development of the maize kernel
   can be divided into three stages: the early development, filling, and
   dehydration stages [[127]1]. In the early development stage (about 0–15
   DAP), the embryo and endosperm cells are quickly divided and
   differentiated into specific tissues. In the filling stage (about 15–45
   DAP), the embryo and endosperm start to accumulate starch and to store
   protein lipids and vitamins [[128]35]. Then, the kernel enters the
   mature stage and begins to dehydrate (about 45–60 DAP) [[129]36].
   Kernel mutants with developmental defects in different stages have been
   characterized, and a great number of genes have been cloned
   [[130]7,[131]8]. These studies have provided valuable information for
   unveiling the gene regulation network of maize kernel development.

   In this study, we characterized a small-kernel mutant smk7a, which
   exhibited arrested embryo and endosperm development compared to the WT.
   The small and pale smk7a kernels with irregular embryo and endosperm
   can be clearly distinguished from the WT at 12 DAP. These results
   suggest that the mutant phenotype of smk7a first occurred before 12
   DAP. This phenotype of smk7a is quite similar to some kernel mutants
   with variant genes related to early maize kernel development. For
   example, the dek15 mutant exhibits a defective endosperm and embryo in
   the early stage, and the embryos appear to be more severely affected
   than the endosperm. The DEK15 encodes cohesin-loading complex subunit
   SCC4, and the loss of the SCC4 function leads to defects in the cell
   division and early kernel development [[132]37]. Both embryo
   development and endosperm development are delayed in the smk4-1 mutant
   compared with that in the WT at 8 DAP. SMK4 encodes an E-subclass PPR
   protein, which is exclusively localized in mitochondria [[133]38]. The
   smk1 mutation leads to defects in the assembly of complex I, which
   results in the arrest of the embryo and endosperm in the early
   development stage [[134]39]. Thus, we speculate that the functional
   gene controlling the smk7a phenotype may play an important role in
   early kernel development.

   Our cytological observations also revealed that the BETL morphology is
   impaired in smk7a. The smk7a BETL developed less-elongated cells and
   had very few cell wall ingrowths. The RNA-seq results revealed that the
   expression levels of several BETL-specific genes were significantly
   downregulated. Defective BETL development usually results in reduced
   sucrose transport from the maternal plant to the endosperm, and it
   ultimately leads to suppressed starch synthesis and endosperm
   development [[135]6,[136]40,[137]41]. For example, ENB1 encodes a
   cellulose synthase 5 that directs the synthesis of cell wall ingrowths
   in maize BETL cells. The enb1 mutant exhibits a drastic reduction in
   the formation of flange cell wall ingrowths in the BETL cells and
   impaired nutrient uptake and starch synthesis [[138]22]. ZmCTLP1
   participates in lipid homeostasis in transfer cells. The loss of the
   function of ZmCTLP1 results in irregular-shaped BETL cells with few
   wall ingrowths and significantly reduced starch, sugar, and protein
   contents in the mutant [[139]4]. Based on our biochemical results, the
   starch content of smk7a was significantly lower, while the soluble
   sugar content was higher in smk7a compared to WT at 12 and 20 DAP.
   Further analysis revealed that the starch accumulation in the WT
   increased from 12 to 20 DAP, which was accompanied by a reduction of
   the soluble sugar content, indicating balanced conversion from soluble
   sugar to starch. In contrast, the soluble sugar content in smk7a
   increased from 12 DAP to 20 DAP. These results suggest that the sugar
   and starch metabolism was disrupted in smk7a.

   The RNA-seq data revealed that at 12 and 20 DAP, the DEGs enriched in
   the “starch and sucrose metabolism” pathway were significantly
   downregulated. Starch began accumulating in the endosperm of the maize
   kernels at approximately 10 DAP, starting with the transport of sucrose
   (Suc) from the maternal plant to the endosperm. Suc was then converted
   into fructose (Fru) and glucose (Glu) by neutral invertase (NINV) or
   into Fru and UDPG by sucrose synthase [[140]42]. The resulting Glu and
   Fru were then phosphorylated to generate hexose phosphates, including
   G6P, F6P, and G1P. G1P was then further activated by AGPase to produce
   ADP-glucose [[141]43], which was the substrate for starch synthesis
   [[142]44,[143]45]. In our RNA-seq results, almost all of the DEGs
   involved in this sucrose metabolism process exhibited a decreased
   expression level in smk7a, including the well-known genes SH1, SH2, and
   BT2. SH1 encodes sucrose synthase, and the loss of the function of SH1
   caused a significant reduction in the carbohydrates that flow to starch
   synthesis, ultimately contributing to the defects in the starch granule
   development and reduction of the starch content [[144]46]. SH2 and BT2
   encode AGPase and are rate-limiting enzymes in starch biosynthesis.
   Mutations in SH2 and BT2 resulted in greatly reduced starch production
   and increased sugar in the endosperm [[145]47,[146]48,[147]49]. In
   smk7a, at 12 DAP, the expressions of SH2 and BT2 were decreased by 9.2-
   and 26-fold, respectively, compared to that in the WT. These results
   explain the increased soluble sugar contents of the smk7a kernels
   compared to that of the WT kernels. After the sugar was metabolized
   into ADP-glucose, starch was synthesized by multiple subunits or
   isoforms of five classes of enzymes, namely, AGP, SS, GBSS, SBE, and
   DBE [[148]33]. In our RNA-seq, the expressions of amylose synthases
   WX1, GBSSI, and GBSSIb; amylopectin synthases SSIV, SSV, and SSI;
   branching enzyme SBE; and starch debranching enzyme SU1 were all
   significantly reduced in smk7a compared to those in the WT. All of the
   above results indicate that the process of starch synthesis is severely
   suppressed in smk7a, which could be the major reason for the defective
   phenotype of the smk7a kernel.

   Our results also revealed that the accumulation of the major storage
   protein zein was significantly reduced in smk7a at 12 and 20 DAP and in
   the mature stage. Consistent with this, all of the zein genes in the
   “nutrient reservoir activity” term (GO: 0045735) were downregulated in
   smk7a. It should be noted that several key regulator factors, including
   O2, O11, and PBF, were also found to be downregulated in smk7a. O2 was
   the first bZIP transcription factor known to regulate the expression of
   zein genes by recognizing the O2 box in their promoters. Loss of the
   function of O2 results in a drastically decreased zein content
   [[149]50]. Several studies subsequently reported that O2 not only acts
   as a master regulator of the zein-coding genes but can also physically
   interact with the prolamin-box binding factor (PBF) to regulate starch
   accumulation [[150]51]. O11 is a central hub of the regulatory network
   for maize endosperm development and nutrient metabolism [[151]52]. It
   works upstream to regulate carbohydrate metabolic enzymes and the key
   regulators (such as O2 and PBF). The decreased expression of these
   genes suggests that the mutation in smk7a has a noticeable effect on
   starch and nutrition accumulation. The candidate gene of smk7a may
   interact with these key regulatory factors and could play an important
   role in the complex regulatory network of maize kernel development.

   IAA is the most abundant endogenous auxin in the maize kernel, and it
   plays an essential role throughout the entire kernel development
   process [[152]53]. In smk7a, the free IAA content was only 58.3% and
   85.0% that of the WT at 12 DAP and 20 DAP, respectively. These results
   suggest that IAA synthesis is suppressed in smk7a. The RNA-seq results
   revealed that the essential genes that participate in the IPA pathway
   for IAA biosynthesis, including ZmYUC1 [[153]54], TAR1, TAR2, and TAR3
   [[154]55,[155]56], are significantly downregulated in smk7a. Defective
   kernel (dek) mutants in maize have been shown to have altered levels of
   IAA. For example, DE18 encodes the auxin biosynthesis gene ZmYUC1, and
   de18 mutation causes a large reduction of the free IAA level compared
   with the WT, leading to a 40% reduction in the dry mass of the
   endosperm. Several studies have also reported that developing kernels
   may regulate growth by influencing the auxin level in response to the
   sugar concentrations. The MN1 gene encodes a cell wall invertase
   [[156]20,[157]57], which is localized in the BETL and catalyzes the
   conversion of sucrose into glucose and fructose. Loss of the function
   of MN1 results in pleiotropic changes, including reduced cell number
   and cell size, a lower kernel mass, and a lower free IAA level
   [[158]58]. In addition, the mn1 mutant also exhibits retarded wall
   ingrowth formation in the BETL cells. Gene expression analysis suggests
   that the expression of the ZmYUC1 and TAR1 genes is greatly reduced in
   mn1 kernels. In smk7a, the BETL cells were impaired and wall ingrowth
   was reduced, which could be the result of disrupted sugar hormone
   signaling in the endosperm.

4. Materials and Methods

4.1. Plant Materials

   In our previous study, a maize small-kernel mutant smk7 was isolated
   from the EMS mutant library produced in our laboratory on the B73
   genetic background [[159]34]. However, its name is repeated with
   another kernel mutant reported in a recent study [[160]59], and
   therefore, we renamed it as smk7a. Since the homozygote smk7a cannot
   grow into a normal plant, the mutants were maintained in heterozygote
   by continuous self-crossing and phenotype selection. The developing
   kernels of WT and smk7a used for cytological, biochemical, and
   transcriptome analysis in this study were collected from the same
   segregating ears of the M8 generation. The heterozygous plant
   (smk7a/WT) was crossed with Mo17 and Chang7-2 and then self-pollinated
   to generate F[2] populations. The segregating F[2] ear were used for
   genetic mapping of smk7a. Maize plants were grown under natural
   conditions at the Changping experimental station of the Institute of
   Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing.

4.2. Light Microscopy and Scanning Electron Microscopy Observations of smk7a
and WT Kernels

   WT and smk7a kernels were acquired from the same heterozygous ear at
   12, 16, 20, 25, and 30 DAP. Then, the kernels were cut along the
   longitudinal axis into half-sections and fixed at room temperature in
   formalin-aceto-alcohol (FAA) solution (90 mL of 70% ethanol, 5 mL of
   glacial acetic acid, and 5 mL of 37% formaldehyde). The fixed material
   was dehydrated in an ethanol gradient series (50%, 70%, 85%, 90%, and
   100% ethanol), embedded in paraffin, and subsequently cut into 12 μm
   sections. The sections were stained with toluidine blue and observed
   under a microscope (BX53; Olympus, Tokyo, Japan).

   The WT and smk7a kernels collected at 34 DAP were fixed in FAA. The
   fixed material was dehydrated in an ethanol gradient series and then
   treated with isoamyl acetate for 15 min twice to replace the remaining
   ethanol. Then, the material was subjected to critical point drying. The
   samples were then coated with Pt particles and analyzed under a
   scanning electron microscope (SU8020, Hitachi, Tokyo, Japan).

4.3. Measurement of Protein and Starch Contents

   Mature WT and smk7a kernels were obtained from the same segregating
   ears at 12 and 20 DAP. After removing the pericarp and embryo, the
   endosperms of the WT and smk7a were each pooled and ground into powder.
   Three biological replicates were prepared for the starch and protein
   measurements. For the starch quantification, 100 mg of powder per
   sample was analyzed using an a-amylase/amyloglucosidase starch assay
   kit (K-TSTA, Megazyme, Ireland) according to the manufacturer’s
   instructions. The starch content of the mature seeds was determined as
   has been previously described by [[161]60]. For the protein
   quantification, 50 mg of powder was used for the protein extraction
   according to the method described by [[162]61]. Then, the protein
   content was measured using a Bradford Protein Assay Kit (PC0010,
   Solarbio, Beijing, China).

4.4. Measurement of Soluble Sugar and Free IAA Contents

   The kernels of WT and smk7a collected at 12 and 20 DAP from the same
   heterozygous ear were ground into power in the presence of liquid
   nitrogen. The soluble sugar and IAA contents were determined by Wuhan
   Greensword Creation Technology Co., Ltd. (Wuhan, China) according to
   previously reported methods [[163]62,[164]63]. The soluble sugars were
   analyzed via ultra-high performance liquid chromatography-electron
   spray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS, Thermo
   Fisher Scientific, Inc., Waltham, MA, USA). The IAA analysis was
   performed using a Thermo Scientific Ultimate 3000 UHPLC system equipped
   with a TSQ Quantiva-Stage Quadrupole Mass Spectrometer (Thermo Fisher
   Scientific, Sunnyvale, CA, USA). Three biological replicates were
   prepared for each measurement.

4.5. RNA Sequencing Analysis

   The total ribonucleic acid (RNA) was isolated from the kernels of WT
   and smk7a collected at 12 and 20 DAP using a RNA Easy Fast Extraction
   Kit (DP452, Tiangen, Beijing, China). A total of 1 μg of RNA per sample
   was used to construct sequencing libraries using a TruSeq Stranded mRNA
   LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the
   manufacturer’s instructions, and index codes were added to attribute
   the sequences to each sample. Then, these libraries were sequenced
   using the Illumina HiSeqTM 2500 platform to generate 125 bp paired-end
   reads. Following the removal of low-quality reads and adaptor
   contaminants, the clean reads were mapped to the maize reference genome
   RefGen_V4
   ([165]ftp.ensemblgenomes.org/pub/plants/release-32/gff3/zea_mays/,
   accessed on 1 November 2018) using HISAT2 [[166]64]. Only perfectly
   matching reads were further analyzed. The gene expression levels were
   estimated using the fragments per kilobase of the transcript per
   million fragments (FPKM) mapped. Differential expression analysis of
   the two groups was performed using DESeq2 [[167]65]. The resulting p
   values were adjusted using Benjamini and Hochberg’s approach for
   controlling the false discovery rate. The genes with an adjusted
   p-value of <0.05 and a fold change of >2 or <0.5 were assigned as
   differentially expressed.

   Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes
   and Genomes (KEGG) enrichment analysis were conducted using R based on
   the hypergeometric distribution, and those with a q value of <0.05 were
   defined as significantly enriched terms or pathways.

4.6. RNA Extraction and Quantitative RT-PCR Verification

   The quantitative real-time polymerase chain reaction (qRT-PCR)
   technique was used to verify the expression level of the differentially
   expressed genes (DEGs) related to the BETL development and starch
   synthesis process. The total RNA of the kernels from the WT and smk7a
   collected at 12 and 20 DAP was extracted using an RNA Easy Fast
   Extraction Kit (DP452, Tiangen, Beijing, China) and then transcribed
   into complementary deoxyribonucleic acid (cDNA) using qPCR RT Master
   Mix with gDNA Remover (KR116, TIANGEN, Beijing, China). The qRT-PCR
   reactions were carried out using Taq Pro Universal SYBR qPCR Master Mix
   (Q712-02, Vazyme, Nanjing, China). The qRT-PCR analysis was performed
   using a Bio-Rad CFX96 qRT-PCR system. The reaction was carried out
   using three biological replicates with three technical replicates, with
   tubulin as the endogenous control (Zm00001d013367). The relative
   expression values were calculated using the 2^−ΔΔCt method [[168]66],
   and the t-test was conducted to analyze the significant differences.
   The specific primers for the qRT-PCR are listed in [169]Table S9.

5. Conclusions

   In summary, we characterized a small-kernel mutant smk7a of maize. Our
   results revealed that: (1) The early development of the embryo and
   endosperm of smk7a is seriously affected. (2) The starch and zein
   protein accumulation is lower in smk7a compared to in WT, and the
   expression levels of the DEGs involved in these processes are
   significantly suppressed in smk7a. (3) The auxin (IAA) content is lower
   in smk7a compared to in WT, and the DEGs that participate in IAA
   synthesis and signaling are downregulated in smk7a. (4) smk7a is a
   recessive gene located at chromosome 2. We speculate that smk7a may be
   related to the sugar and hormone signaling in the maize kernel and may
   play a crucial role in embryo and endosperm development.

Acknowledgments