Abstract

   Improving grain yield in the staple food crop rice has been long sought
   goal of plant biotechnology. One of the traits with significant impact
   on rice breeding programs is peduncle elongation at the time of heading
   failing which leads to significant reduction in grain yield due to
   incomplete panicle exsertion. To decipher transcriptional dynamics and
   molecular players underlying peduncle elongation, we performed RNA
   sequencing analysis of elongating and non-elongating peduncles in two
   Indian cultivars, Swarna and Pokkali, at the time of heading. Along
   with genes associated with cell division and cell wall biosynthesis, we
   observed significant enrichment of genes associated with auxins,
   gibberellins, and brassinosteroid biosynthesis/signaling in the
   elongating peduncles before heading in both the genotypes. Similarly,
   genes associated with carbohydrate metabolism and mobilization, abiotic
   stress response along with cytokinin, abscisic acid, jasmonic acid, and
   ethylene biosynthesis/signaling were enriched in non-elongating
   peduncles post heading. Significant enrichment of genes belonging to
   key transcription factor families highlights their specialized roles in
   peduncle elongation and grain filling before and after heading,
   respectively. A comparison with anther/pollen development-related genes
   provided 76 candidates with overlapping roles in anther/pollen
   development and peduncle elongation. Some of these are important for
   carbohydrate remobilization to the developing grains. These can be
   engineered to combat with incomplete panicle exsertion in male sterile
   lines and manipulate carbohydrate dynamics in grasses. Overall, this
   study provides baseline information about potential target genes for
   engineering peduncle elongation with implications on plant height,
   biomass composition and grain yields in rice.

   Keywords: heading, rice, peduncle, stem, anther, panicle exsertion,
   internode

Introduction

   With huge diversity in morphological architectures of flowering plants,
   a thorough understanding of plant morphology is not only essential to
   appreciate the diverse shapes and forms exhibited by the plant world
   but also vital to engineering crop plants with improved agricultural
   traits. A slight modification in plant architecture can have a profound
   effect on the agronomic performance of crop plants. This was clearly
   demonstrated during the green revolution, where breeding for semi-dwarf
   genotypes led to higher yields and lodging resistance in rice
   ([35]Coyne, 1980). Subsequently, several architectural features such as
   shape, size and angle of the leaf, number of tillers, and branching,
   were found to dramatically impact grain yield and stress responses
   ([36]Peng et al., 2008).

   One of the crucial morphological traits that significantly influences
   grain yield in rice is the elongation of the uppermost internode, also
   known as the peduncle. Elongation of peduncle at the time of heading
   facilitates the emergence of panicle from the flag leaf sheath,
   commonly known as panicle exsertion, which is also crucial for anther
   dehiscence and pollination ([37]Bardenas, 1965). Incomplete panicle
   exsertion dramatically reduces seed set in rice and thus adversely
   affects agronomic yield ([38]Yin et al., 2007). Moreover, peduncle
   elongation is highly sensitive to environmental factors. Several
   abiotic stress factors, particularly extremes of temperature and
   drought stress at the time of heading, result in dramatic yield losses
   by inhibiting peduncle elongation and, subsequent panicle exsertion and
   anther dehiscence in rice ([39]Muthurajan et al., 2011; [40]He and
   Serraj, 2012; [41]Wu et al., 2016). Incomplete panicle exsertion is
   also observed in cytoplasmic male sterile lines with about 30–40% of
   panicles remaining enclosed in the flag leaf sheath, therefore making
   the spikelets unavailable for cross-pollination ([42]Devi et al.,
   2011). Thus, incomplete panicle exsertion is one of the major
   impediments to obtain high yields in rice breeding programs. One of the
   common strategies used to combat this challenge involve spray of
   gibberellins but it significantly enhances the cost of production
   ([43]Yin et al., 2007).

   Furthermore, peduncle also contributes to grain filling. The excess
   photoassimilates, accumulated in the form of sucrose and starch in rice
   stems before heading, are mobilized to the panicles after heading to
   supplement grain filling ([44]Chen and Wang, 2008; [45]Wang et al.,
   2016). The transport of these non-structural carbohydrates from flag
   leaf to the panicle is facilitated through a continuous mature
   transport phloem network in leaf sheath, peduncle, and the rachis that
   supports spikelets on panicle ([46]Slewinski, 2012). Since peduncle
   goes through a sink to source transition during the heading stage,
   elucidating transcriptional dynamics of peduncle at the time of heading
   is of fundamental importance to understand the mechanism of
   non-structural carbohydrate remobilization as well.

   Till date, a handful of genes have been experimentally characterized to
   affect internode elongation through forward and/or reverse genetic
   strategies ([47]Rutger and Carnahan, 1981; [48]Luo et al., 2006;
   [49]Zhu et al., 2011; [50]Duan et al., 2012; [51]Gao et al., 2012;
   [52]Magome et al., 2013; [53]Ji et al., 2014, [54]2019; [55]Wang et
   al., 2017; [56]Xie et al., 2018; [57]Chu et al., 2019). However, lack
   of a comprehensive study to understand the overall gene expression
   dynamics during panicle elongation at the time heading is still
   limiting our understanding of panicle exsertion process in rice.

   To fill this gap in our understanding, we investigated the
   transcriptional dynamics during peduncle elongation at the time of
   heading in two indica genotypes of rice, Swarna and Pokkali, so that
   genotype-specific effects could be minimized. These genotypes exhibit
   contrasting physiological and morphological traits in terms of cell
   wall composition, plant height, and grain yields ([58]Jahn et al.,
   2011). Swarna, an elite indica cultivar, developed by crossing Vasista
   and Mahsuri rice varieties in 1982, has low glycemic index and is the
   most widely grown rice cultivar in Southern India ([59]Rathinasabapathi
   et al., 2015). Pokkali, on the other hand, is a salinity tolerant
   indica landrace, mainly grown in coastal region of Southern India, for
   its salinity tolerance, high protein content, extra-large grains,
   peculiar taste, and medicinal properties ([60]Hoang et al., 2016;
   [61]Mishra et al., 2020). Expression profiling of elongating and
   non-elongating peduncles collected before and after heading,
   respectively, from both the genotypes, revealed conserved genetic
   elements and pathways underlying peduncle elongation at the time of
   heading in rice. These genes can be targeted using reverse genetic
   approaches to increase yield stability and stress tolerance in rice
   ([62]Slewinski, 2012). Those implicated in cell wall biosynthesis would
   be important candidates for engineering stem biomass composition to
   enhance biofuel production. At the same time, genes regulating anther
   development as well as peduncle elongation can be leveraged to devise
   strategies to combat panicle enclosure in male sterile lines.

Materials and Methods

Plant Material and Sample Preparation

   Oryza sativa ssp. indica cultivars Swarna and Pokkali plants were grown
   in the fields under puddle transplanted conditions at ICAR-Indian
   Agricultural Research Institute (ICAR-IARI), Pusa, New Delhi, India.
   The phenotypic data on plant height, peduncle length, days to heading,
   and maturity were recorded from the field-grown plants. Elongating
   peduncles (EP) about 2–4 days before heading, and non-elongating
   peduncles (NP), about 2–4 days after heading, were separately sampled
   for each cultivar. Before heading, samples were collected when the
   maximum bulge could be observed in the flag leaf with the panicle still
   concealed beneath the leaf sheath. Conversely, heading stage samples
   were collected once the panicle had fully emerged out from the flag
   leaf sheath accompanied by flowering ([63]Figure 1A). The samples were
   frozen in liquid nitrogen and stored at −80°C until further processing.

FIGURE 1.

   [64]FIGURE 1
   [65]Open in a new tab

   Phenotypic assessment and transcriptomic analysis of rice peduncles.
   (A) The topmost internode, also known as peduncle, is marked in both
   before heading and after heading Pokkali and Swarna plants. All leaves
   were removed to clearly show the stem internodes. (B) Graph showing
   comparative length of elongating and non-elongating peduncles in Swarna
   and Pokkali plants. (C) Principal component analysis of all three
   biological replicates of each sample. Dots of the same color represent
   biological replicates from same sample. (D) Venn diagram showing
   overlap between number of genes expressing with FPKM ≥ 1 in each
   sample. The total number of genes expressed in each sample is given in
   brackets with sample names. SW, Swarna; PK, Pokkali; EP, elongating
   peduncles before heading, and NP, non-elongating peduncles after
   heading.

RNA Isolation, Library Preparation, and Sequencing

   Total RNA from three biological replicates of peduncle samples,
   collected from each cultivar at both elongating and non-elongating
   stages, was extracted using TRIzol^TM reagent (Invitrogen) as per the
   manufacturer’s instructions. The quantity and quality of RNA samples
   were determined using Nanodrop 2000 and Agilent 2100 Bioanalyzer. RNA
   samples with RIN value >7 were processed for library preparation using
   TruSeq^® Stranded Total RNA Sample Preparation Kit. Sequencing was
   performed on Hisequation 2000 with a read length of 100 bp (Illumina).
   The data has been submitted to NCBI GEO (Accession No. [66]GSE157727).

Data Analysis

   After sequencing, raw reads were filtered to remove low-quality and
   adapter-contaminated reads using NGSQC Toolkit with default parameters
   ([67]Patel and Jain, 2012). The high-quality reads with Phred score ≥30
   were aligned with the rice genome available at Rice Genome Annotation
   Project Database version 7^[68]1, using Hisat version 2.1.0 with the
   option – rna-strandness RF, specific for stranded reads ([69]Kim et
   al., 2015). Assembly was performed using Stringtie with −rf option
   ([70]Pertea et al., 2015). The final assembly for all samples was
   obtained by Cuffmerge ([71]Trapnell et al., 2012). Differential gene
   expression between both the samples was determined by Cuffdiff using
   the – fr option ([72]Trapnell et al., 2012). The transcripts with
   log[2]fold change ≥1 (upregulated genes) or ≤−1 (downregulated genes)
   with P-value cutoff ≤0.05 were considered differentially expressed. The
   expression levels of novel and annotated genes were quantified in terms
   of Fragments Per Kilobase per Million (FPKM) using Cufflinks
   ([73]Trapnell et al., 2010). Principal component analysis (PCA) was
   performed on normalized FPKM values using prcomp function (center =
   TRUE, scale = FALSE) of the R package stats^[74]2. The 3D diagram of
   PCA for all 12 samples was plotted using the R package scatterplot3d
   (version 0.3–37^[75]3). Gene ontology (GO) enrichment for
   differentially expressed transcripts was performed using
   PlantGSEA^[76]4; ([77]Yi et al., 2013), and P-values for enrichment
   analysis of each GO term were calculated using F-test. Only GO terms
   exhibiting P-value ≤0.05 were considered significant.

   For metabolic pathway analysis, differentially expressed genes were
   also mapped on Mapman software using mapping file available for IRGSP
   loci ([78]Thimm et al., 2004;^[79]5). The information about previously
   characterized genes for their roles in internode elongation,
   anther/pollen development, and grain filling was collected from OGRO
   (Overview of functionally characterized Genes in Rice Online database;
   [80]Yamamoto et al., 2012;^[81]6) and FunRice genes ([82]Yao et al.,
   2018;^[83]7) databases supplemented with manual literature survey. The
   list of differentially expressed genes in nine rice male sterile
   mutants was extracted from supplementary data provided by [84]Lin et
   al. (2017).

Results

Plant Growth and Phenotypic Analysis

   The plant height and developmental stages of Oryza sativa spp. indica
   genotypes, Swarna and Pokkali, grown in natural fields, were monitored
   over time especially closer to the heading stage. Both heading and
   maturity take longer in Pokkali as compared to Swarna plants as Pokkali
   plants took on an average of 123 and 151 days after sowing for heading
   and maturity, respectively, whereas, in Swarna plants, the average
   numbers of days for heading and maturity were 112 and 136 days after
   sowing, respectively. Phenotypic data collected from field-grown plants
   revealed striking contrast in plant height and peduncle length in both
   the genotypes ([85]Figure 1A). The average height of Pokkali was almost
   double (158 cm) compared to Swarna (82 cm) plants. A similar trend was
   observed in the peduncle length. The average length of elongating
   peduncles 2–4 days before heading was 2.9 and 5.3 cm in Swarna and
   Pokkali, respectively. However, with prolific elongation observed
   closer to heading, peduncle lengths increased to an average of ∼20 cm
   in Swarna and ∼30 cm in Pokkali plants within 2–4 days post heading
   ([86]Figure 1B). No significant increase in peduncle length was
   observed 4 days post heading, suggesting that the peduncle ceases to
   elongate as soon as the whole panicle has emerged out of the flag leaf.

Sequencing Statistics and Principal Component Analysis

   RNA sequencing of 12 libraries representing elongating (EP) and
   non-elongating peduncles (NP) of both the genotypes yielded a total of
   690 million paired-end reads with an average read length of 100 bp.
   After removing low-quality and adapter contaminated reads, >616 million
   high-quality reads with an average Phred quality score (Q) ≥30 were
   used for reference-based transcriptome assembly. More than 90% of the
   high-quality reads from each replicate could be mapped onto the rice
   genome ([87]Supplementary Table 1), and a total of 54,966 transcripts
   were annotated from both the cultivars.

   To emphasize variation and correlation among EP and NP stages collected
   from both the genotypes, we performed PCA. As expected, elongating
   peduncles exhibited a distinct separation from the non-elongating ones
   ([88]Figure 1C). Furthermore, the results revealed a higher correlation
   among EP samples of both the cultivars. In contrast, non-elongating
   peduncles from both the cultivars were well-separated indicating
   genotypic differences in the transcriptional repertoire of
   non-elongating peduncles ([89]Figure 1C).

   Further, genes expressed with FPKM ≥ 1 in all four samples were
   compared ([90]Figure 1D). The total number of genes expressing in EP
   and NP stages in both the genotypes were comparable with more than
   16,000 genes expressing at EP stage and over 15,000 genes expressing in
   NP samples from both the genotypes. A total of 12,726 transcripts were
   common to both EP and NP samples. In both the genotypes, Swarna had
   higher number of genes expressing exclusively in elongating (609) as
   well as non-elongating (841) peduncles ([91]Figure 1D).

In silico Validation of Gene Expression Patterns

   Several genes in rice have been previously implicated in peduncle
   elongation and panicle exsertion at the time of heading using forward
   and/or reverse genetic approaches. To investigate if we could capture
   their differential accumulation during peduncle elongation, we checked
   the expression patterns of nine previously characterized genes in our
   data ([92]Figure 2). Among these, OsPK1 encoding a pyruvate kinase has
   been previously implicated in regulating plant height, panicle
   exsertion and carbohydrate transport during grain filling ([93]Zhang et
   al., 2012). The mutant exhibits dwarfism, panicle enclosure, and
   reduced seed set. Consistent with its demonstrated role, OsPK1
   expressed at high levels in elongating peduncles before heading which
   escalated further in non-elongating peduncles post heading in both the
   genotypes ([94]Figure 2). Another gene, OsSUT1, encoding the sucrose
   transporter, has been previously shown to express in rice peduncle with
   a critical role in facilitating the transport of assimilates from the
   flag leaf blade to the base of filling grains ([95]Scofield et al.,
   2002, [96]2007). We observed a significant increase in OsSUT1
   expression post-heading in peduncles of both the genotypes ([97]Figure
   2).

FIGURE 2.

   [98]FIGURE 2
   [99]Open in a new tab

   Expression profiles of previously characterized genes for involvement
   in peduncle elongation and/or panicle exsertion. The Y-axis represents
   FPKM values, and the X-axis represents samples. Error bars represent
   standard error between biological replicates. SW, Swarna; PK, Pokkali;
   EP, elongating peduncles, and NP, non-elongating peduncles. The names
   of the genes are given in italics on the top left of each bar graph.

   A cytochrome P450 gene, CYP714B1, that encodes gibberellin 13-oxidase
   has been shown to reduce gibberellin (GA) activity in rice ([100]Magome
   et al., 2013). Double mutation in CYP714B1 and CYP714B2 resulted in
   elongation of uppermost internode in rice at the time of heading
   indicating their role in suppressing internode elongation post heading
   by downregulating GA activity. Conforming to these observations,
   CYP714B1, with negligible expression before heading, showed high
   expression in rice peduncles after heading in both the rice genotypes
   ([101]Figure 2), whereas expression of a GA biosynthetic gene, GA3Ox2
   (Gibberellin 3 beta-dioxygenase 2) that plays a decisive role in
   internode elongation in rice, was detected in elongating peduncles
   pre-heading in both the genotypes ([102]Iwamoto et al., 2011;
   [103]Figure 2).

   Dwarfism 50 (D50) encoding inositol polyphosphate 5-phosphatase is
   essential for proper formation of intercalary meristem by regulating
   the direction of cell division, deposition of cell wall pectin, and
   actin organization ([104]Sato-Izawa et al., 2012). As expected, D50
   exhibited higher expression in rice peduncles before heading with a
   significant decline observed post heading in both the genotypes
   ([105]Figure 2). Similarly, another gene, SUI1 (Shortened Uppermost
   Internode 1) encoding phosphatidylserine synthase, mediates cell
   expansion during elongation of uppermost internode in rice by
   regulating the secretion of cell wall components ([106]Zhu et al.,
   2011; [107]Yin et al., 2013; [108]Ma et al., 2016). The sui1 mutants
   exhibit immensely shortened uppermost internode and a sheathed panicle
   indicating its role in peduncle elongation and panicle exsertion at the
   time of heading. SUI1, though expressed at very low levels, exhibited a
   significant decline in expression post heading in both the genotypes
   ([109]Figure 2).

   Furthermore, rice genes associated with brassinosteroid biosynthesis
   and signaling have also been shown to affect internode elongation. Loss
   of function of BRD2 (Brassinosteroid-deficient dwarf 2) involved in the
   brassinosteroid biosynthetic pathway and a putative BR receptor kinase,
   BRI1 (Brassinosteroid Insensitive 1), leads to dwarf phenotypes
   confirming their role in internode elongation ([110]Yamamuro et al.,
   2000; [111]Hong et al., 2005). Both the genes exhibited a higher
   expression in elongating peduncles before heading in rice genotypes
   with a significant decline post heading in our data. Similarly,
   phytochrome-regulated OsEREBP1-like transcription factor EBL1 which
   regulates internode elongation at the heading stage in rice by
   upregulating the expression of ACO1 (aminocyclopropane-1-carboxylate
   oxidase), an enzyme associated with ethylene biosynthesis ([112]Iwamoto
   and Takano, 2011), exhibited a significant decline in expression in
   Pokkali plants post heading ([113]Figure 2). On the contrary, EBL1
   expression in Swarna plants was negligible both before and after
   heading. The differential expression of EBL1 could be due to
   differential photoperiod sensitivity or response to ethylene levels in
   both the genotypes. Overall, we obtained the expected expression
   patterns of the previously characterized genes conforming to their
   demonstrated functions.

Differential Gene Expression Analysis and Pathway Enrichment

   To identify genes exhibiting differential accumulation during peduncle
   elongation at the time of heading in both the genotypes, we used a
   cutoff ≥2-fold change with P-value ≤0.05. Expression levels of 8,894
   and 5,018 genes were altered in Swarna and Pokkali, respectively. A
   total of 4,280 and 2,348 genes were expressed at higher levels in
   elongating peduncles of Swarna and Pokkali, respectively ([114]Figure
   3A), whereas 4,614 and 2,670 genes expressed at higher levels in
   non-elongating peduncles of Swarna and Pokkali, respectively. Since our
   study aimed to identify the genes with conserved roles in peduncle
   elongation instead of genotype-specific differences, we compared the
   differentially expressed gene sets from both the genotypes. A total of
   1,500 genes were expressed at higher levels in EPs of both the
   genotypes compared to NPs ([115]Figure 3B). Similarly, 1,723 genes
   expressed at higher levels in non-elongating peduncles. A large number
   of genes exhibiting contrasting transcript profiles imply
   genotype-specific variations likely responsible for variation in cell
   wall, biomass composition, stress tolerance, and other physiological
   parameters ([116]Figure 3B).

FIGURE 3.

   [117]FIGURE 3
   [118]Open in a new tab

   Results of differential expression analysis and pathway mapping. (A)
   Bar diagram showing the number of genes differentially expressed (fold
   change ≥ 2 and P-value ≤ 0.05) at the time of heading with EP
   representing the number of genes exhibiting higher expression in
   elongating peduncles before heading and NP representing number of genes
   exhibiting higher expression post heading. SW, Swarna; PK, Pokkali. (B)
   Venn diagrams showing the overlap between differentially expressed
   genes in both the genotypes. (C) Pathway enrichment analysis of the
   differentially expressed genes, with EP representing genes exhibiting
   higher expression in elongating peduncles before heading and NP
   representing genes exhibiting higher expression in non-elongating
   peduncles post heading, in both the genotypes. The size of square
   correlates with number of genes in each category while color signifies
   P-values as shown by legend on the right.

   To identify critical genes and pathways underlying peduncle elongation,
   irrespective of the genotype, we performed pathway enrichment analysis
   with 1,500 and 1,723 genes exhibiting differential expression in
   peduncles in both the genotypes ([119]Supplementary Table 2). Among
   1,500 genes with higher expression in elongating peduncles, those
   involved in transcriptional regulation, posttranslational
   modifications, cell organization, DNA synthesis, cell wall
   biosynthesis, lipid metabolism, stress response, cell cycle, and auxin
   metabolism were particularly enriched ([120]Figure 3C). On the other
   hand, the enriched categories among 1,723 genes exhibiting higher
   expression in non-elongating peduncles were transcriptional regulation,
   protein degradation, posttranslational modification, development,
   calcium signaling, stress response, amino acid metabolism, sugar
   transport, and ethylene metabolism ([121]Figure 3C). These observations
   conform with the developmental events involving active cell division in
   the elongating peduncles and mobilization of carbohydrates in
   non-elongating peduncles after heading. Among those exclusively
   upregulated in elongating peduncles of Swarna, protein synthesis, RNA
   processing, development, and photosynthetic light reaction categories
   were enriched. On the contrary, genes associated with vesicular
   transport, signaling, and cell wall biosynthesis were exclusively
   enriched in elongating peduncles of Pokkali ([122]Supplementary Table
   5). Interestingly, genes associated with photosynthesis were
   exclusively upregulated in non-elongating peduncles of Pokkali, whereas
   those associated with protein degradation, posttranslational
   modification, signaling, lipid metabolism, and protein targeting were
   exclusively enriched in non-elongating peduncles of Swarna plants
   ([123]Supplementary Table 5).

In-Depth Analysis of Differentially Expressed Genes Associated With
Transcriptional Regulation, Cell Wall, Carbohydrate, and Hormone Metabolism

   Taking cues from the pathways enriched during peduncle elongation, we
   performed an in-depth profiling of differentially expressed genes
   associated with cell wall biosynthesis and modification, carbohydrate
   metabolism and transport, hormone biosynthesis, signaling, and
   response, and transcriptional regulation.

Cell Wall Biosynthesis and Modification

   A total of 24 cellulose synthases (CESA) and cellulose synthase-like
   (CSL) genes were differentially expressed in both the genotypes with 22
   of them exhibiting higher expression in elongating peduncles compared
   to non-elongating ones ([124]Figure 4A; [125]Supplementary Table 3A).
   The CSL genes exhibiting higher expression in elongating peduncles
   belong to the CSLA, CSLC, CSLC, CSLD, and CSLF families ([126]Figure
   4A). CSLE6 and CSLH1, on the contrary, exhibited higher expression post
   heading ([127]Figure 4A). Several genes implicated in secondary
   metabolite biosynthesis also exhibited higher expression in post
   heading peduncles ([128]Figure 4B).

FIGURE 4.

   [129]FIGURE 4
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   Expression profiling of genes associated with cell wall biosynthesis
   and secondary metabolism. (A) Heatmap showing differential expression
   of cell wall biosynthesis-related genes during heading in Swarna (SW)
   and Pokkali (PK) plants. Teal blue color represents genes exhibiting
   ≥2-fold expression in elongating peduncles while cayenne red indicates
   ≥2-fold expression in non-elongating peduncles post heading. Gene names
   are given on the right of the heatmap. (B) MapMan secondary metabolism
   overview with green boxes presenting genes with higher expression in
   elongating peduncles and red boxes representing genes exhibiting higher
   expression in non-elongating peduncles. (C) Heatmap showing
   differential expression of lignin biosynthesis-related genes. Teal blue
   color represents genes exhibiting ≥2-fold expression in elongating
   peduncles while cayenne red indicates ≥2-fold expression in
   non-elongating peduncles post heading. Gene names are given on the
   right of the heatmap.

   However, genes associated with lignin biosynthesis showed a mixed
   pattern with PAL (phenylalanine ammonia-lyase) and CCR (cinnamoyl-CoA
   reductase) genes expressing at higher levels in EPs while 4CL
   (4-coumarate: coenzyme A ligase) and CCOMT (caffeoyl CoA
   3-O-methyltransferase) exhibited higher expression in non-elongating
   peduncles ([131]Figure 4C; [132]Supplementary Table 3B). Further, three
   genes encoding 4CLs (4-coumarate: coenzyme A ligases) and COMT (caffeic
   acid 3-O-methyltransferase) exhibited contrasting patterns in both the
   genotypes with higher expression in elongating peduncles of Pokkali and
   non-elongating peduncles of Swarna rice. These results may be explained
   by varying levels of lignification of peduncles after heading in both
   the genotypes.

Carbohydrate Metabolism and Transport

   Since the peduncle comprises an essential component of the transport
   system facilitating carbohydrate mobilization during grain filling post
   heading, we investigated the expression of rice genes implicated in
   sucrose and starch metabolism as well as transport in our data
   ([133]Supplementary Table 3C). Several genes regulating sucrose and
   starch degradation, as well as sugar transport, exhibited differential
   expression in peduncles at the time of heading ([134]Figure 5). Notable
   among those were sucrose synthases ([135]Huber and Akazawa, 1986;
   [136]Stein and Granot, 2019). Rice SUS4 (sucrose synthase 4), which has
   previously been shown to be associated with grain filling exhibited
   higher expression in post heading peduncles, whereas SUS1 (sucrose
   synthase 1) having a more ubiquitous role was downregulated after
   heading in peduncles ([137]Figure 5A). Antagonistic patterns of these
   genes are likely due to their differential activity in different
   tissues. SUS1 is implicated in the regulation of vegetative growth,
   while SUS4 is involved in assimilate portioning to the caryopsis during
   grain filling ([138]Sparks(ed.), 2012).

FIGURE 5.

   [139]FIGURE 5
   [140]Open in a new tab

   Expression profiling of genes associated with carbohydrate metabolism
   and transport. The outer illustration is the MapMan transport overview
   with green boxes presenting genes with higher expression in elongating
   peduncles and red boxes representing genes exhibiting higher expression
   in non-elongating peduncles. The inner portion presents heatmaps
   showing differential regulation of genes associated with sucrose
   metabolism (A), starch metabolism (B), and sugar transport (C) Swarna
   (SW) and Pokkali (PK) plants. Teal blue color represents ≥2 folds
   expression in elongating peduncles and cayenne red indicates ≥2 folds
   expression in non-elongating peduncles post heading. Gene names are
   given on the right of each heatmap.

   Invertases modulate the hexose to sucrose ratio by hydrolyzing sucrose
   into glucose and fructose. Most of the invertases exhibited a higher
   expression in post heading peduncles ([141]Figure 5A) Both cytosolic
   invertase 1 (CIN1) and vacuolar invertase (INV3) were upregulated after
   heading ([142]Morey et al., 2018). A similar trend was observed for
   other starch metabolism and sugar transport-related genes with
   significant upregulation after heading ([143]Figure 5B).

   Before loading into the sieve element–companion cell complex through
   sucrose transporters, sucrose is effluxed from phloem parenchyma cells
   by SWEET (Sugars Will Eventually Be Exported Transporters) genes
   ([144]Ayre, 2011; [145]Chen et al., 2012). We observed significant
   upregulation of several SWEET genes in rice peduncles after heading
   ([146]Figure 5C). Besides, the genes encoding ABC, potassium, calcium,
   peptides, amino acids, nucleotides, phosphate, sulfate, ammonium,
   nitrate, and P- and v-ATPase transporters also exhibited a higher
   expression post heading. Several genes associated with carbohydrate
   partitioning such as SUT1 and GWD1 also exhibited higher expression in
   peduncles after heading ([147]Figure 5C). The sucrose
   transporter-encoding gene OsSUT1 has previously been shown to play a
   role in long-distance transport of assimilates from the flag leaf blade
   to the base of filling grains through the flag leaf sheath and
   uppermost internode, referred to as peduncle in this study
   ([148]Scofield et al., 2002, [149]2007). GWD1, encoding alpha-glucan
   water dikinase, has been implicated in carbohydrate partitioning after
   heading in rice ([150]Wada et al., 2017). Loss of function of GWD1 led
   to hyperaccumulation of starch in leaves ([151]Hirose et al., 2013).

Hormone biosynthesis, signaling, and response

   Among differentially expressed genes, 68 genes have been previously
   implicated in plant hormone biosynthesis/signaling ([152]Figure 6;
   [153]Supplementary Table 3D). Although some of them showed a
   contrasting pattern, by and large genes associated with auxins and
   brassinosteroids (BR) exhibited higher expression in elongating
   peduncles before heading, whereas those regulating abscisic acid (ABA),
   jasmonic acid (JA), and cytokinin metabolism were predominantly
   upregulated post heading ([154]Figure 6). The genes associated with GA
   and ethylene biosynthesis/signaling and response exhibited a mixed
   pattern though overall trend suggested higher expression of larger
   number of genes post heading ([155]Figure 6).

FIGURE 6.

   [156]FIGURE 6
   [157]Open in a new tab

   Expression profiling of genes associated with hormone biosynthesis and
   signaling. Heatmaps showing differential expression of genes associated
   with hormone biosynthesis and signaling in Swarna (SW) and Pokkali
   (PK). Teal blue color represents ≥2 folds expression in elongating
   peduncles and cayenne red indicates ≥2 folds expression in
   non-elongating peduncles post heading. Gene names are given on the
   right of the heatmaps and hormone categories are given on the left.

   Similarly, although majority of ABA-biosynthesis/signaling genes
   expressed at higher levels in post-heading peduncles, some exhibited
   contrasting expression patterns in both genotypes. Several of the ABA
   signaling-related genes which exhibited a higher expression after
   heading in peduncles of Swarna, expressed at higher levels before
   heading in Pokkali plants ([158]Figure 6). Conversely, some of the
   genes associated with ABA deactivation that expressed at higher levels
   before heading in Swarna were upregulated in peduncles of Pokkali.
   These observations point to ABA activation in Swarna while ABA
   deactivation in Pokkali after heading.

Transcription Factor-Encoding Genes

   A total of 836 transcription factors (TFs), belonging to more than
   twenty families, were differentially expressed in rice peduncles after
   heading ([159]Supplementary Table 3E). Albeit members of the same
   transcription factor family do not necessarily perform the same
   function, we observed a large number of genes belonging to the same
   family exhibiting similar expression patterns, thereby indicating
   similar/coordinated functions of the gene family members. Genes
   belonging to ARF, AUX-IAA, GRF, HMG, OFP, Trihelix, PHD, and TCP
   families, mostly implicated in cell division and elongation, exhibited
   a higher expression in elongating peduncles ([160]Figure 7), whereas
   those belonging to ARR-B, C2C2-CO like C2C2-Dof, MADS, TRAF, AP2-EREBP,
   bZIP, NAC, G2-like, HSF, OPHANS, WRKY, TIFFY, REP-RK, and PLATZ
   families expressed at higher levels post heading. Members of C3H, SNF2,
   C2H2, bHLH, GRAS, and HB exhibited mixed patterns ([161]Figure 7).

FIGURE 7.

   [162]FIGURE 7
   [163]Open in a new tab

   Expression profiling of transcription factor-encoding genes. Heatmaps
   showing differential expression of transcription factor-encoding genes
   in Swarna (SW) and Pokkali (PK). Members of the same gene family are
   clubbed together in one heatmap. Teal blue color represents ≥2 fold
   expression in elongating peduncles while cayenne red indicates ≥2 fold
   expression in non-elongating peduncles post heading. Names of the
   transcription factor families are given on the right of each heatmap.

Differential Expression of Anther/Pollen-Associated Genes During Peduncle
Elongation

   Incomplete peduncle elongation and panicle enclosure are frequently
   observed in male sterile lines. To investigate if the genes associated
   with peduncle elongation have anything to do with anther development,
   we compared the differentially expressed genes during peduncle
   elongation with those associated with anther/pollen development.
   Previously, [164]Lin et al. (2017) had generated a rice gene
   co-expression network for anther development (RiceAntherNet) from 57
   rice anther tissue microarrays. They mapped differentially expressed
   genes from nine rice male-sterile mutants onto this network and
   shortlisted a set of 286 genes associated with pollen formation. We
   appended this list with anther/pollen-associated genes extracted from
   Funrice and OGRO databases of rice. Finally, a list of 493 genes
   implicated in anther/pollen development in rice was compiled. Out of
   these 493 genes, 76 (15%) were differentially expressed in peduncles at
   the time of heading in both the genotypes ([165]Figure 8;
   [166]Supplementary Table 4). Out of these, 50 overlapped with genes
   exhibiting ≥2-fold expression in EPs, whereas 26 overlapped with genes
   exhibiting ≥2-fold expression in NPs ([167]Figure 8). Most of the
   anther development-related genes that exhibited higher expression in
   elongating peduncles before heading (50 in number) encode glycoside
   hydrolases, serine carboxypeptidases, kinases, and other cell
   wall-related proteins. Some of these genes have already been
   characterized for their roles in regulating plant height, grain filling
   and/or anther/pollen development. Conversely, those overlapping with
   high-expressing genes in non-elongating peduncles (26 in number) as
   well as anther development have been implicated in abiotic stress and
   grain filling.

FIGURE 8.

   [168]FIGURE 8
   [169]Open in a new tab

   Overlap between differentially expressed genes during peduncle
   elongation and anther development. The Venn diagram shows the overlap
   between genes exhibiting ≥2 fold expression in elongating peduncles
   (EP), non-elongating peduncles (NP), and anther/pollen development. The
   key pathways and characterized genes highlighted in the gene set common
   between EP/NP and anther/pollen associated genes are given.

Discussion

   Peduncle elongation at the time of heading not only is crucial for
   panicle exsertion but also exerts significant impact on grain yield. To
   identify molecular players regulating peduncle elongation and
   associated agronomically important traits, we have performed RNA
   sequencing with peduncles before and after heading collected from two
   contrasting genotypes of rice. Although the total number of expressed
   genes (FPKM ≥1) was similar in both the genotypes, those exhibiting
   differential accumulation between EP and NP stages in Swarna were
   almost double in number compared to differentially expressed genes in
   Pokkali. This could possibly be because Swarna has to make more
   transcriptional adjustments to cope up with the abiotic stress post
   heading compared to the relatively stress-tolerant Pokkali. Further,
   most of the genes exclusively differentially expressed in Swarna are
   involved in the basic metabolic functions including protein synthesis,
   degradation and posttranslational modifications, signaling, RNA
   processing, lipid metabolism, transport, development, and abiotic
   stress response. PCA analysis further highlighted noticeable distance
   between non-elongating peduncles compared to elongating peduncles
   likely due to differences in post heading stem biomass composition and
   stress response of these genotypes ([170]Jahn et al., 2011). However,
   when we compared the expression patterns of previously characterized
   genes which have been experimentally shown to regulate peduncle
   elongation and grain filling in rice, all of them exhibited a similar
   expression pattern in both the genotypes though the amplitude of
   expression and fold change varied, indicating that the genetic factors
   underlying peduncle elongation are conserved across genotypes.
   Furthermore, changes observed at the transcriptional level may not
   reflect in protein abundance. Earlier, comparative analysis of
   transcript and protein abundance of Pokkali and IR29 revealed a much
   higher number of genes differentially expressed in Pokkali at the
   protein level with little difference at the transcript level in
   response to salt stress ([171]Li et al., 2018). In fact, 75.5% of genes
   exhibiting high protein abundance did not show relatively high
   transcriptional abundance in Pokkali. The authors suggested that higher
   stability and efficient loading of mRNAs in Pokkali could be the
   possible explanation for lower number of differentially expressed
   transcripts in Pokkali.

Candidate Genes Associated With Peduncle Elongation and Grain Filling

   Elongation of peduncles is accompanied by active cell division and cell
   wall biosynthesis. This is obvious from enrichment of
   glycosyltransferase (GTs) in elongating peduncles of both the
   genotypes. Glycosyltransferases comprise a large gene family with more
   than 600 genes in rice which have been classified into subfamilies
   based on the presence of distinct sequence domains ([172]Cao et al.,
   2008). Among these, CESAs belonging to the GT2 subfamily are mainly
   involved in cellulose biosynthesis, while cellulose synthase-like (CSL)
   genes play key roles in hemicellulose biosynthesis ([173]Richmond and
   Somerville, 2001). CSLAs form the β-1,4-mannan backbone of
   galactomannan and glucomannan, CSLDs determine cellulose and xylose
   content, whereas, CSLFs are essential for grass-specific mixed linkage
   glucan biosynthesis ([174]Li et al., 2009; [175]Luan et al., 2011;
   [176]Vega-Sánchez et al., 2012; [177]Kaur et al., 2017). Many of these
   have been shown to exhibit tissue and developmental stage-specific
   expression. In-depth analysis of these genes in our data provided 22
   candidates with significant accumulation in EPs relative to NPs
   implying their potential role in regulating cell wall biosynthesis and
   biomass composition in elongating rice peduncles.

   Also, a shift from primary to secondary cell wall biosynthesis is
   anticipated at the time of heading for providing mechanical strength
   required to support increase in panicle weight due to grain filling.
   OsMYB103 has earlier been reported to play a role in secondary cell
   wall in sclerenchyma by activating OsCESA4, OsCESA7, OsCESA9, and OsBC1
   ([178]Hirano et al., 2013; [179]Yang et al., 2014; [180]Ye et al.,
   2015). All of these genes exhibit higher accumulation in EPs indicating
   that molecular players responsible for secondary cell wall biosynthesis
   are initiated before heading. On the other hand, higher accumulation of
   genes associated with biosynthesis of secondary metabolites in NPs
   indicates their role in post-heading resistance to pathogens though
   some of them may also act as precursors for secondary cell wall
   components. Genes involved in lignin biosynthesis exhibited contrasting
   pattern of expression in Swarna and Pokkali likely due to varied lignin
   composition in these genotypes ([181]Jahn et al., 2011).

   Among the differentially expressed transcription factor families,
   members of MYB, NAC, WRKY, bZIP, PLATZ, and HSF transcription factors
   have been previously associated with secondary growth, abiotic stress
   response, and carbon mobilization ([182]Oh et al., 2003; [183]Agarwal
   et al., 2006; [184]Zhang et al., 2010; [185]Golldack et al., 2014).
   Further characterization of these genes would not only help to decipher
   the molecular mechanism underlying peduncle elongation and biomass
   composition but will also help in optimizing carbon remobilization in
   rice.

Interplay of Phytohormones During Peduncle Elongation in Rice

   Phytohormones play crucial roles in plant growth and development.
   Previous studies have shown the involvement of auxins, gibberellins,
   and brassinosteroids in promoting cell division and growth during
   internode elongation in rice ([186]Chen, 2001; [187]Yin et al., 2002;
   [188]Tong et al., 2014; [189]Oh et al., 2020). For instance, BR
   receptor kinase OsBRI1 of rice positively regulates internode
   elongation by inducing the formation of the intercalary meristems
   ([190]Yamamuro et al., 2000). Loss of function of this gene prevents
   internode elongation and bending of the lamina joint. We noticed a high
   expression of OsBRI1 before heading in both the genotypes with an
   apparent decline after heading. Except for OsBRL3, which is known to
   play a role in BR perception in roots ([191]Nakamura et al., 2006), all
   other brassinosteroid-related genes also exhibited a higher expression
   in elongating peduncles before heading. Similarly, auxins also act as a
   signal for cell elongation ([192]Chen, 2001). Developing panicles in
   grasses export auxin to stem for promoting elongation ([193]Wolbang et
   al., 2004). Except for a few, most of the auxin-related genes were
   expressed at higher levels in rice peduncles before heading. Among the
   genes exhibiting higher expression post heading, OsARF19 is a negative
   regulator of cell division as its overexpression in rice leads to dwarf
   phenotype. Furthermore, OsARF19 directly regulates the expression of BR
   receptor OsBRI1 by binding to its regulatory region ([194]Zhang et al.,
   2015). Upregulation of OsARF19 and downregulation of OsBRI1 after
   heading therefore suggest that OsARF19 likely suppresses peduncle
   elongation after heading by downregulating OsBRI1.

   Gibberellic acid (GA) has also been shown to act as a key determining
   factor in peduncle elongation and panicle exsertion ([195]Yin et al.,
   2007; [196]Chen et al., 2013). We observed a mixed pattern of the genes
   associated with GA and ethylene biosynthesis/signaling likely due to
   multilevel cross-talk or negative feedback regulation of some of them.
   GA biosynthetic gene, gibberellin 3-beta-hydroxylase (GA3Ox), which
   catalyzes the final step of GA biosynthesis in plants by converting
   GA20 to GA1, exhibited a higher expression before heading with notable
   downregulation after heading ([197]Iwamoto et al., 2011; [198]Figure
   6). OsGA3Ox2 corresponds to the D18 mutant that exhibits severe
   dwarfism due to decrease in level of active GA1 ([199]Itoh et al.,
   2002). Antisense plants with reduced OsGA3Ox2 expression exhibited a
   higher expression of another gene OsGA20Ox1 that regulates the
   synthesis of GA21 from GA53 ([200]Itoh et al., 2002). Interestingly,
   both the genes in our study exhibited antagonistic patterns with GA3Ox2
   exhibiting downregulation and GA20Ox1 showing upregulation after
   heading. Conversely, GA receptor Gibberellin Insensitive Dwarf 1 (GID1)
   which makes a complex with GA and induces degradation of GA repressing
   DELLA proteins is upregulated ([201]Ueguchi-Tanaka et al., 2007;
   [202]Sun, 2008) while DELLA protein-encoding genes, SLR1 (SLENDER RICE
   1) and SLR1 Like-1 (SLRL1), are downregulated after heading, indicating
   that suppression of GA activity after heading is not mediated through
   DELLA proteins. It has been shown that a high concentration of BR
   triggers BR-associated inactivation of GA. The BR signaling component
   involved in this inactivation process, BRI1-GSK2-DLT, is upregulated
   before heading in our data ([203]Tong et al., 2014). The effect of this
   can be observed with increased expression of GA deactivation genes
   after heading. For instance, CYP714B1 encoding gibberellin 13-oxidase
   which reduces gibberellin activity in rice as well as GA2ox4 and
   GA2ox6, implicated in endogenous GA deactivation ([204]Chu et al.,
   2019), expressed at higher levels in peduncles after heading ([205]Lo
   et al., 2008; [206]Magome et al., 2013). Overall, regulation of OsBRI1
   by OsARF19 and its (OsBRI1) role in deactivation of GA indicates
   interaction of auxin, brassinosteroids, and gibberellins in maintaining
   the hormonal homeostasis during peduncle elongation ([207]Zhang et al.,
   2015).

   Cytokinins, on the other hand, have been associated with post-anthesis
   grain filling in rice ([208]Yang et al., 2002; [209]Zhang et al.,
   2009). Since mostly cytokinins are transported from roots to shoots and
   other aerial organs via xylem sap, elevated levels of cytokinins in
   rice peduncles after heading suggest their role in grain filling
   ([210]Yang et al., 2002; [211]Zhang et al., 2009). Panicle enclosure
   has been shown to adversely affect spikelet fertility and grain yield
   ([212]He and Serraj, 2012). A genome-wide association study using 205
   rice cultivars clearly drew a positive correlation between panicle
   exsertion and 1,000-Grain Weight ([213]Zhan et al., 2019). The grain
   filling is facilitated by transport of carbohydrate resources stored in
   elongating stem in rice in the form of starch and sucrose
   ([214]Scofield et al., 2007, [215]2009; [216]Slewinski, 2012). The
   carbon remobilization from the stem reserves to grains possibly occurs
   by an apoplasmic mechanism through sugar transporters ([217]Slewinski,
   2012). Many of the sugar transport genes were upregulated in the
   peduncles suggesting their role in grain filling. For example, OsSUT1,
   a member of the sucrose transporter family, is required for actively
   pumping sucrose into phloem against the concentration gradient
   ([218]Scofield et al., 2007). In the present study, its upregulation
   before heading conforms with its role in grain filling. Monosaccharide
   transporter genes, OsMST4 and 6, implicated in supplying
   monosaccharides for seed development during grain filling, are also
   upregulated in NPs after heading in our study ([219]Wang et al., 2007,
   [220]2008). OsSUS4, involved in portioning of the carbon assimilates to
   the caryopsis during grain filling ([221]Sparks(ed.), 2012), is also
   upregulated in peduncles after heading. This suggests that peduncle
   plays a critical role in transporting carbohydrate reserves stored
   during stem elongation to the developing grains, and hence, rice genes
   exhibiting increased expression in NPs after heading may be leveraged
   to optimize carbohydrate mobilization in rice.

   Jasmonic acid (JA) is another important phytohormone associated with
   biotic and abiotic stress response and is known to inhibit shoot growth
   in rice explaining the upregulation of JA biosynthetic genes after
   heading ([222]Liu et al., 2015). The level of JAs has also been
   associated with availability of carbon and soluble sugars, indicating
   the role of JA in carbon remobilization ([223]Huang et al., 2017).
   Upregulation of ABA-related genes after heading has been associated
   with abiotic stress response during reproductive development
   ([224]Baron et al., 2012; [225]Singh et al., 2012). Some genes
   implicated in ABA biosynthesis and signaling exhibited contrasting
   patterns of expression in both the genotypes. Further investigations
   would be required to understand if this can be explained by
   differential stress tolerance in Swarna and Pokkali post heading.
   Overall, our data shows a clear trend with higher expression of growth
   hormones, viz., AUX, GA, and BR, in elongating peduncles before
   heading, whereas JA, ABA, and cytokinins, associated with stress
   response, sugar signaling, and remobilization, exhibit higher
   expression after heading.

Common Genetic Elements Underlying Peduncle Elongation and Male Reproductive
Organ Development

   Crop improvement in rice to optimize agronomic yields and stress
   tolerance largely relies on breeding programs. However, panicle
   enclosure due to impeded peduncle elongation in male sterile lines is a
   major challenge in achieving these goals. A number of studies have
   associated enclosed spikelets with inhibition of anther dehiscence,
   decreased pollen viability, slower pollen tube growth, and abnormal
   ovary development ([226]Ekanayake et al., 1989, [227]1990;
   [228]Jagadish et al., 2011, [229]2015). A comparative study of
   cytoplasmic male sterile line with its maintainer lines during panicle
   development revealed increased levels of ABA and reduced levels of GA
   and IAA in the cytoplasmic male sterile line ([230]Chang’en et al.,
   1998). Deficiency of IAA and GAs and excess of ABA hinder anther
   development and induce pollen abortion ([231]Shimizu and Kuno, 1967;
   [232]Nakajima et al., 1991; [233]Sunohara et al., 2009). Later,
   [234]Yin et al. (2007) showed that deficiency of indole-3-acetic acid
   in panicle of male sterile lines downregulates GA oxidase (OsGA3ox2)
   resulting in decreased active GA levels, thereby hampering peduncle
   elongation manifested in the form of enclosed panicles and poor seed
   set ([235]Yin et al., 2007). In the present study, several genes
   related to anther and pollen development were differentially expressed
   in both elongating and non-elongating peduncles. Although it is a
   well-known fact that male sterile lines exhibit varying degrees of
   panicle enclosure, the genetic basis of the relationship between anther
   development and peduncle elongation has not been established till date.
   To identify the common genetic elements regulating peduncle elongation
   and anther/pollen development, we compiled a list of 493 genes
   implicated in anther/pollen development and checked their expression in
   peduncles.

   Most of the anther development-related genes, also exhibiting higher
   expression in elongating peduncles before heading (50 in number),
   encode glycoside hydrolases, serine carboxypeptidases, and kinases.
   Some of these genes have already been characterized for their roles in
   anther development and/or internode elongation. Among these, DCW11
   ([236]Fujii and Toriyama, 2008), OsSTRL2 [strictosidine synthase;
   ([237]Zou et al., 2017)], EDT1 [Earlier Degraded Tapetum 1; ([238]Bai
   et al., 2019)], OsABCG3 [ATP-binding cassette transporter; ([239]Chang
   et al., 2018)], OsGH3.8 ([240]Yadav et al., 2011), OsSPO11D
   ([241]Shingu et al., 2012), OsUAM3 ([242]Sumiyoshi et al., 2015), and
   OsUCL23 ([243]Zhang et al., 2020) have been shown to regulate diverse
   aspects of anther and pollen development. OsSTRL2 expression was
   detected only in tapetal cells and microspores, and knockout mutant
   resulted in male sterility mainly due to defects in anther wall and
   pollen exine formation ([244]Zou et al., 2017). Several others in this
   category have been shown to regulate both vegetative and reproductive
   organ development. For example, DNL1 (Dwarf and Narrow Leafed 1)
   encoding cellulose synthase like D4 is the major QTL regulating plant
   height and leaf width in rice. The mutant exhibits defects in anther
   dehiscence and pollen formation as well ([245]Yoshikawa et al., 2013).
   Similarly, antisense transgenic plants of OsTUB8 encoding β-tubulin
   exhibit shorter plant height as well as reduced seed set ([246]Yang et
   al., 2007). Loss of function of OsER2 (ERECTA) encoding phytosulfokine
   receptor led to reduced plant height and panicle size by affecting cell
   proliferation and growth in rice ([247]Zhang et al., 2018).
   Furthermore, SLG (Slender Grain) from this group encodes BAHD
   acyltransferase and has been shown to regulate leaf angle, grain
   length, and plant height by modulating brassinosteroid levels ([248]Liu
   et al., 2016). Interestingly, OsSWEET11, involved in sucrose transport
   and maintaining the sucrose concentration in the embryo-sac, also
   exhibited higher expression in EPs before heading ([249]Ma et al.,
   2017).

   Similarly, some of the genes exhibiting high-expressing genes in
   non-elongating peduncles as well as anther development have been
   experimentally shown to play role in grain filling and abiotic stress
   response. Among these, OsMGD2 (monogalactosyldiacylglycerol synthase)
   affects plant height, anther development, and overall rice grain yield
   ([250]Basnet et al., 2019). SSG6 (Substandard Starch Grain 6) has
   previously been shown to regulate starch grain morphology and size in
   pollen and seeds ([251]Matsushima et al., 2016). The ssg6 mutant
   affected duration to heading, culm length, number of panicles, and seed
   weight. OsSUT1 an important sucrose transporter is also upregulated
   after heading. Disruption of OsSUT1 does not affect pollen maturation
   but their function gets impaired ([252]Hirose et al., 2010), whereas
   OsMST6 ([253]Monfared et al., 2020) and OsNHX1 ([254]Almeida et al.,
   2017) regulate abiotic stress response. Interestingly, OsCIPK23
   (calcineurin B-like interacting protein kinase) has been implicated in
   mediating common signaling pathways between pollination and drought
   stress response ([255]Yang et al., 2008).

   The pivotal roles played by these genes in regulating anther
   development, plant height, grain filling, and abiotic stress response
   at the time of heading suggest that these as crucial candidates for
   alleviating the problem of panicle enclosure in male sterile lines and
   related agronomically important traits.

Data Availability Statement

   The original contributions presented in the study are publicly
   available. This data can be found here:
   [256]https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE157727.

Author Contributions

   RS conceived and designed the study. MK performed all the experiments
   and the data analysis. MK and RS drafted the manuscript. CV, VC, KK,
   AP, and MKS participated in experiment design, sampling as well as
   implementation of the project. All authors read and approved the
   manuscript.

Conflict of Interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

   Funding. This study was carried out with the financial support from JNU
   through UPE-II and DST (Department of Science and Technology)-Purse-II
   grants. RS and MKS acknowledge Department of Biotechnology, Government
   of India, for Ramalingaswami fellowships. MK thanks JNU and UGC for
   non-NET fellowship. KK acknowledges University Grants Commission (UGC),
   Government of India, for Rajiv Gandhi National Fellowship. Financial
   assistance from Science and Engineering Research Board (SERB),
   Department of Science and Technology (DST), Government of India
   (ECR/2016/001581), to RS and MKS is also duly acknowledged.
   ^1

   [257]http://rice.plantbiology.msu.edu/
   ^2

   [258]https://stat.ethz.ch/R-manual/R-devel/library/stats/html/stats-pac
   kage.html
   ^3

   [259]https://cran.r-roject.org/web/packages/scatterplot3d/index.html
   ^4

   [260]http://systemsbiology.cau.edu.cn/PlantGSEA/
   ^5

   [261]https://mapman.gabipd.org/
   ^6

   [262]http://qtaro.abr.affrc.go.jp/ogro
   ^7

   [263]https://funricegenes.github.io/

Supplementary Material

   The Supplementary Material for this article can be found online at:
   [264]https://www.frontiersin.org/articles/10.3389/fgene.2020.584678/ful
   l#supplementary-material
   Supplementary Table 1

   Summary statistics of RNA sequencing.
   [265]Click here for additional data file.^ (9.9KB, XLSX)
   Supplementary Table 2A

   List of genes exhibiting higher expression in non-elongating peduncles
   (NP) compared to elongating peduncles (EP) in both Swarna and Pokkali.
   [266]Click here for additional data file.^ (328.3KB, XLSX)
   Supplementary Table 2B

   List of genes exhibiting higher expression in elongating peduncles (EP)
   compared to non-elongating peduncles (NP) in both Swarna and Pokkali.
   [267]Click here for additional data file.^ (328.3KB, XLSX)
   Supplementary Table 3A

   List of differentially expressed cellulose synthase and synthase like
   genes in rice peduncles.
   [268]Click here for additional data file.^ (82.5KB, XLSX)
   Supplementary Table 3B

   List of differentially expressed lignin biosynthesis genes in rice
   peduncles.
   [269]Click here for additional data file.^ (82.5KB, XLSX)
   Supplementary Table 3C

   List of differentially expressed carbohydrate metabolism and
   transport-related genes in rice peduncles.
   [270]Click here for additional data file.^ (82.5KB, XLSX)
   Supplementary Table 3D

   List of differentially expressed hormone-related genes in rice
   peduncles.
   [271]Click here for additional data file.^ (82.5KB, XLSX)
   Supplementary Table 3E

   List of differentially expressed transcription factor encoding genes in
   rice peduncles.
   [272]Click here for additional data file.^ (82.5KB, XLSX)
   Supplementary Table 4

   List of differentially expressed genes in rice peduncles overlapping
   with anther/pollen associated genes extracted from RiceAntherNet,
   funRiceGenes and OGRO databases.
   [273]Click here for additional data file.^ (21.2MB, XLSX)
   Supplementary Table 5A

   Enriched MapMan pathways among upregulated genes in elongating
   peduncles (EP) of both Pokkali and Swarna plants.
   [274]Click here for additional data file.^ (21.9KB, XLSX)
   Supplementary Table 5B

   Enriched MapMan pathways among the genes exclusively upregulated in
   elongating peduncles (EP) of Pokkali.
   [275]Click here for additional data file.^ (21.9KB, XLSX)
   Supplementary Table 5C

   Enriched MapMan pathways among exclusively upregulated genes in
   elongating peduncles (EP) of Swarna.
   [276]Click here for additional data file.^ (21.9KB, XLSX)
   Supplementary Table 5D

   Enriched MapMan pathways among upregulated genes in non-elongating (NP)
   peduncles of both Pokkali and Swarna plants.
   [277]Click here for additional data file.^ (21.9KB, XLSX)
   Supplementary Table 5E

   Enriched MapMan pathways among exclusively upregulated genes in
   non-elongating (NP) peduncles of Pokkali.
   [278]Click here for additional data file.^ (21.9KB, XLSX)
   Supplementary Table 5F

   Enriched MapMan pathways among exclusively upregulated genes in
   non-elongating (NP) peduncles of Swarna.
   [279]Click here for additional data file.^ (21.9KB, XLSX)

References