Abstract

   The alteration of heavy (“ON/bearing”) and light (“OFF/non-bearing”)
   yield in pistachio (Pistacia vera L.) has been reported to result from
   the abscission of inflorescence buds on high yielding trees during the
   summer, but the regulatory mechanisms involved in this bud abscission
   remain unclear. The analysis provides insights into the transcript
   changes between inflorescence buds on bearing and non-bearing shoots,
   that we indicated as “ON” and “OFF”, and shed light on the molecular
   mechanisms causing premature inflorescence bud abscission in the
   pistachio cultivar “Bianca” which can be related to the alternate
   bearing behavior. In this study, a transcriptome analysis was performed
   in inflorescence buds of “ON” and “OFF” shoots. A total of 14,330
   differentially expressed genes (DEGs), most of which are involved in
   sugar metabolism, plant hormone pathways, secondary metabolism and
   oxidative stress pathway, were identified. Our results shed light on
   the molecular mechanisms underlying inflorescence bud abscission in
   pistachio and we proposed a hypothetical model behind the molecular
   mechanism causing this abscission in “ON” shoots. Results highlighted
   how changes in genes expressed in nutrient pathways (carbohydrates and
   mineral elements) in pistachio “ON” vs. “OFF” inflorescence buds
   triggers a cascade of events involving trehalose-6-phosphate and target
   of rapamycin (TOR) signaling, SnRK1 complex, hormones, polyamines and
   ROS which end, through programmed cell death and autophagy phenomena,
   with the abscission of inflorescence buds. This is the first study
   reporting gene expression profiling of the fate of “ON” and “OFF”
   inflorescence buds associated with the alternate bearing in the
   pistachio.

   Keywords: Pistacia vera, polyamines, flower bud abscission, alternate
   bearing

1. Introduction

   Pistachio (P. vera L.) originates in the arid areas of central Asia, in
   the areas of the Caspian Sea (Iran) and the territories between
   Afghanistan and Kyrgyzstan [[36]1]. The pistachio is a wind-pollinated
   deciduous, dioecious tree which presents cyclic variation of fruiting,
   usually of two years, in which heavy production occurs during the “ON”
   year and less/no production in the following “OFF” year. The mechanism
   regulating the alternate bearing phenomenon in pistachio is unique
   [[37]1,[38]2]. In a mature pistachio tree, all the main vegetative and
   reproductive phases are carried out by the plant in a short period,
   between the mid of March and the end of May. The growth pattern of the
   current season’s shoot is exclusively dominant, and it extends from the
   vegetative terminal bud of the previous season’s shoot. Under each of
   the compound leaf on the current season’s growth, there is a single
   axillary bud. Most of these axillary buds differentiate into
   inflorescence primordia; therefore, flowering and fruit production
   occurs on 1-year-old wood [[39]2,[40]3,[41]4].

   Thus, unlike other alternate bearing crop species, pistachio produces
   floral buds on current-year shoot but, in the “ON” year, inflorescence
   buds start to detach starting from the basal end of the current-year
   shoot and then towards the apical end [[42]2]. Meanwhile, on the
   contrary, there is bud retention in low crop load (“OFF”) years. Bud
   abscission is considered the visible mechanism underlining the
   alternate bearing [[43]2].

   In order to simplify this phenomenon by outlining a time-line, lower
   buds start to abscise or drop at the end of June and continues in July
   and August, determining the heavy reduction in production in the next
   year, thus resulting in an “OFF” year. The figure below shows the
   growth pattern of the shoot during the “ON” and “OFF” season
   ([44]Figure 1).

Figure 1.

   [45]Figure 1
   [46]Open in a new tab

   (A): Fruit clusters on one-year-old wood and lateral inflorescence buds
   on current year’s bearing shoot of P. vera L. indicated by yellow
   circles (June “ON”); (B): one-year-old wood and lateral inflorescence
   buds on current year’s not-bearing shoot (July “OFF”); (C): Red circles
   show the sites of inflorescence bud abscission in current year’s
   bearing shoot (July “ON”).

   The physiological mechanism, which triggers the inflorescence buds drop
   linked to the alternate bearing behavior in pistachio, is not
   completely clear and two hypotheses are considered, one of which
   involves nutritional factors and the other involves hormonal factors.
   On the basis of the nutritional hypothesis, the competition of the
   growing embryos with the new inflorescence buds for the use of
   metabolites, carbohydrates and nitrogen can be the main cause of
   inflorescence bud dropping [[47]5,[48]6]. The hormonal hypothesis
   suggests that some growth regulators are directly involved in bud
   abscission. However, subsequent studies conducted on the levels of
   abscisic acid (ABA) in fruits and inflorescence buds did not show any
   relationship between the levels of this hormone and the bud drop
   [[49]2,[50]7].

   The nutritional theory suggests that the inflorescence bud drop occurs
   in coincidence with the period of embryo growth and is more intense
   when the crop on one-year-old shoot is heavy, since the embryo
   represents the strongest “sink” [[51]8]. This temporal coincidence of
   bud drops, and nut development suggests a competition between the
   developing embryo and inflorescence buds for the available resources.
   The lack of competitive ability of inflorescence buds compared to fruit
   in attracting the photosynthates produced by the leaves was
   demonstrated by tracking the translocation of the radioactive carbon
   isotope, C14 [[52]7]. It also confirmed that in branches that are
   subjected to annular decortications, also known as shoot girdling
   (removal of a bark ring from the base of the current year’s shoot to
   separate it from the fruitescences), it is possible to reduce the
   inflorescence bud drop by 70% [[53]9]. Similar results emerged from a
   study on the accumulation of nitrogen, phosphorus and potassium on the
   various organs of the branch, showing that the inflorescence buds of
   non-bearing branches accumulated significantly greater quantities of
   macro elements, compared to the inflorescence buds of the bearing
   branches [[54]10].

   Various studies have also highlighted the direct correlation between
   the fruit load and the intensity of the drop of the inflorescence buds
   [[55]1,[56]2,[57]3,[58]5,[59]11]. Studies proved that the presence of
   the infructescence decrease the growth of the leaves and of the shoot
   axis [[60]11,[61]12], and that plants deprived of the fruits for the
   next years accumulate more carbohydrates and thus they express a
   greater potential for growth compared to those left in the normal year
   production cycle [[62]13]. Many studies showed significant changes in
   starch content and the difference in translocation of starch in tissues
   of “ON” and “OFF” trees [[63]2,[64]8,[65]9,[66]11,[67]12,[68]14].

   Moreover, in pistachio, within the canopies of “OFF” trees, it is
   possible to find some “ON” shoots and within canopies of “ON” trees,
   there always some “OFF” shoots; therefore, the theory of shoot or
   branch autonomy should be considered [[69]2,[70]7]. Shoot autonomy in
   fruit trees depends on resource (carbon, water, nutrients and hormone
   distribution) availability. These results underline the importance of
   reserve substances which, although stored in the permanent organs of
   the plant during the “OFF” year, are not enough for the full expression
   of the vegetative growth potential and the fructification. Interesting
   results have emerged from the study of the influence of polyamines
   (putrescine, spermine and spermidine) on the inflorescence bud drops
   [[71]15]. In general, the level of polyamines is negatively correlated
   with inflorescence bud drops.

   Genetic mechanisms involved in alternate bearing have been recently
   studied by transcriptomic analyses in some fruit crops, such as apple
   [[72]16], citrus [[73]17] and olive [[74]18], where the alternate
   bearing is explained as the lack of flower bud initiation and their
   morphological differentiation, unlike in pistachio. In citrus, the
   fruit load critically affects bud fate before that flower induction
   occurs and an alternate bearing signal may be generated in the fruit or
   in another organ that perceives the flowering initiation and the change
   of key metabolic pathways [[75]17]. It has been demonstrated in many
   fruit crops that “ON” and “OFF” crop status is associated with changes
   in the expression of flowering control genes [[76]16,[77]18]. Genes
   regulating trehalose and flavonoid metabolism and genes homologous to
   Squamosa promoter binding-like (SPL) were found induced in “OFF” buds
   of citrus [[78]17].

   In apple, microarray analysis showed that flower induction genes were
   differentially regulated between “ON” and “OFF” inflorescence buds and
   critical changes occur in expression of genes involved in oxidative
   stress, cell wall biogenesis, carbohydrate biosynthesis and lipid
   metabolism [[79]16]. In olive, a cDNA library experiment performed on
   different developmental stages of leaves and fruits in “ON” and “OFF”
   trees showed that P450 monooxygenase and two dehydrins were more
   expressed in leaves of “ON” trees than in leaves of “OFF” trees
   [[80]16]. Furthermore, in “ON” olive trees, a UDP-glucose epimerase, an
   acyl-CoA binding protein, a triose phosphate isomerase and a putative
   nuclear core anchor protein were more expressed in fruits. In “ON” and
   “OFF” olive trees, differences in miRNA-targeted genes were also found
   involved in main hormone signal-transduction pathways and carbohydrate
   metabolism which can be potentially associated in alternate bearing
   processes [[81]18].

   Preliminary transcriptional analysis in pistachio based in only a year
   of observation showed that in inflorescence buds of “ON” bearing
   shoots, photosynthesis related genes were down-regulated and some
   terpenoids related genes were up-regulated [[82]19]. This study can be
   considered as the first study reporting and documenting the gene
   expression profiling associated with inflorescence bud abscission.
   Studies proved that genes associated with bud abscission may involve in
   alternate bearing directly or indirectly [[83]2]. The aim of this
   analysis is to provide insights into the transcript changes between
   inflorescence buds in bearing and non-bearing shoots in order to
   identify the molecular mechanism causing premature inflorescence bud
   abscission, which is linked to alternate bearing in the Italian
   pistachio cultivar “Bianca”. Our results demonstrate the nutritional
   theory and the involvement of a complex network of hormonal signals and
   cross talk in the inflorescence bud drops of fruiting shoots. These
   findings have important implications for the horticultural management
   of this fruit species and can help in the breeding strategies for
   selecting the parents to cross the pistachio tree.

2. Materials and Methods

2.1. Plant Material, RNA Extraction, Processing and Sequencing

   The transcriptomic analysis was conducted taking the tissue samples
   from one mature P. vera (L.) tree of the cultivar “Bianca”, grown
   inland of Sicily (37°30′ Lat. N), in 27th of June 2018 and in 22nd of
   July 2019. The inflorescence buds from bearing (“ON”) and non-bearing
   (“OFF”) branches were analyzed. Bearing branches showed from 40 to 50
   fruits; non-bearing had no fruits. We collected 4–6 inflorescence buds
   each from three branches (considered as three biological replicates) of
   the same tree during the “ON” and “OFF” status of June and July which
   constitute a total of 12 samples. All bud samples were immediately
   frozen in liquid nitrogen after collection and stored at −80 °C. The
   samples were grounded in liquid nitrogen and total RNA extraction was
   performed with the Spectrum^TM Plant Total RNA Kit (Sigma-Aldrich,
   Milan, Italy) employing 100 mg of frozen tissue. RNA quality and RNA
   Integrity Number (RIN) were checked by using the Bioanalyzer. Libraries
   were obtained using the TruSeq RNA-Seq sample prep kit from Illumina
   (Illumina, Inc., San Diego, CA, USA). The 12 samples were loaded into
   one lane of an Illumina flow cell, and clusters were created by
   Illumina Bot. The clusters were sequenced using the service provided by
   BMR Genomics (Padua, Italy) at ultra-high throughput on the Illumina
   HiSeq 2000 (Illumina Inc.) to obtain single reads per sample, each 75
   bp long.

2.2. De Novo Assembly, Evaluation and Annotation

   The quality of the raw sequences generated from transcriptome
   sequencing was assessed with FastQC (version 1.16)
   ([84]https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). With
   respect to the FastQC report, the low-quality bases (Q-score < 30) were
   removed using custom made Perl script and the adaptor sequences were
   removed using cutadapt (version 2.0). The filtered reads were then
   aligned against Silva database ([85]https://www.arb-silva.de/) using
   bowtie [[86]20] (version 2.3.4.1) in order to remove rRNA reads and to
   obtain clean reads. The total pre-processed reads from all 12 samples
   were then de novo assembled using Trinity [[87]21] (version 2.8.4)
   using default parameters. The transcripts from Trinity assembly were
   further clustered using CD-Hit-EST [[88]22] (version 4.6.8), using a
   clustering threshold of 98% identity to reduce redundancy.

   The assembly statistics were obtained using the transrate [[89]23]
   ([90]http://hibberdlab.com/transrate/) program. The assembly was
   evaluated with BUSCO [[91]24] (version 3.0.2), a tool that assesses
   genome completeness based on the presence of single-copy orthologs,
   using the green plant dataset (viridiplantae_odb10). The complete
   assembly statistics and evaluation statistics is given in [92]Table S1.
   The complete workflow of the Pistachio de novo transcriptome assembly
   and annotation were summarized in [93]Figure S1.

   The obtained contigs were annotated using BLASTx program
   ([94]http://www.ncbi.nlm.nih.gov/BLAST/) with an E-value threshold of 1
   × 10^−5 to NCBI nr database
   ([95]https://www.ncbi.nlm.nih.gov/refseq/about/nonredundantproteins),
   UniProt protein database ([96]https://www.uniprot.org), InterPro
   database ([97]https://www.ebi.ac.uk/interpro/), KEGG database
   ([98]http://www.genome.jp/kegg), PFAM database
   ([99]https://pfam.xfam.org) and STRING database
   ([100]https://string-db.org). We considered only the contigs
   corresponds to ‘Viridiplantae’ and the unannotated contigs for the
   final transcriptome assembly. RNA-Seq data were deposited in NCBI’s
   sequence read archive (SRA) under accession number PRJNA623387.

2.3. Differentially Expressed Genes (DEG) between Stages

   To estimate the expression levels of the Trinity reconstructed
   transcripts, we used RSEM [[101]25]. RSEM is a package used to estimate
   the gene and isoform expression levels from RNA sequence data. The
   expected count matrix derived from RSEM is given as the input for edgeR
   [[102]26]. The comparison selected for the study is given in [103]Table
   1. Genes represented with an adjusted p-value (FDR) lower than 0.01 and
   at least a two-fold change were only considered as significantly
   differentially expressed in the pairwise comparison of the samples. In
   addition, the functional-enrichment analysis was performed to identify
   which gene ontology (GO) terms and metabolic pathways that were
   significantly enriched in differentially expressed genes (DEGs).

Table 1.

   The number of total genes, up-regulated and down-regulated genes in
   inflorescence buds in current year non-fruiting shoot “OFF” and in
   inflorescence buds in fruiting shoots “ON”.
   Comparison (Inflorescence Bud) Differentially Expressed Genes
   Up-Regulated Down-Regulated
   July “OFF” vs. July “ON” 1087 247 840
   June “OFF” vs. July “OFF” 2299 976 1323
   June “ON” vs. July “OFF” 2450 591 1859
   June “OFF” vs. July “ON” 2768 820 1948
   June “ON” vs. July “ON” 3882 712 3170
   June “OFF” vs. June “ON” 1844 1409 435
   [104]Open in a new tab

2.4. Gene Enrichment and Functional Analysis

   The final contigs were aligned against TAIR10
   ([105]https://www.arabidopsis.org) protein sequence using blastx
   program, in order to get the corresponding TAIR Id. The blastx result
   files were parsed and generated a Pistachio mapping file for Mapman
   containing the five categories (a) Nearly identical: Score ≥ 1000 and
   e-value = 0 (b) Highly similar: Score ≥ 1000 and e-value ≠ 0 OR (Score
   ≥ 500 & Score < 1000) and e-value = 0 (c) Moderately similar: (Score ≥
   200 & Score < 1000) and e-value ! = 0 (d) Weakly similar: (Score ≥ 100
   & score < 200) (e) Very weakly similar: (Score < 100) based on the
   blastx score and e-value. The MapMan mapping file of Pistachio is
   available from
   [106]https://drive.google.com/file/d/1nMp2euy36JwVtXIrVibEdnkaVB_AV2Uv/
   view.

   We used MapMan ([107]http://mapman.gabipd.org/) [[108]27] with the
   Pistachio mapping file to map the gene IDs and visualize the metabolic
   overview, hormone regulation, CHO metabolism, secondary metabolism and
   transcription factors using two generated files: (1) related to “ON”
   and “OFF” stages of bud, (2) related to two time-point (June and July).

   The PageMan analysis plugin of MapMan was used to visualize differences
   among metabolic pathways using Wilcoxon tests, no correction, and an
   over-representation analysis (ORA) cutoff value of 3. We considered all
   the differentially expressed genes present that are related to the
   comparison of “ON” and “OFF”, June and July for the PageMan analysis.

   The TAIR IDs were searched against the Database for Annotation,
   Visualization and Integrated Discovery (DAVID) version 6.8 [[109]28]
   Web server ([110]https://david.ncifcrf.gov/). The gene ontology
   information related to the biological process was extracted from the
   DAVID result.

3. Results

3.1. De Novo Transcriptome Assembly and Annotation

   High-throughput sequencing technology has provided an excellent
   opportunity for transcriptome survey in non-model plant species,
   including P. vera. To examine the inflorescence bud abscission
   phenomenon of P. vera relate to alternate bearing, inflorescence buds
   from three separate shoots of the same tree were collected and
   sequenced from bearing and non-bearing shoots. The picking dates of the
   material coincide with a period of the initial competition between
   fruit and inflorescence buds, not causing inflorescence bud drop, and a
   period of strong completion, causing the drop of inflorescence buds.

   The sequencing of the data of June produced 199 million raw reads (60
   Gb of data), whereas July produced 196 million reads (59 Gb of data) as
   a single-end. The high-quality single-end reads with an average quality
   score of 38 were selected for the transcriptome assembly after trimming
   off the low-quality bases and adapters from the June and July data sets
   ([111]Table S2). The total pre-processed reads were then de novo
   assembled using Trinity and transcripts from Trinity assembly were
   further clustered using CD-Hit-EST. The assembly was evaluated with
   BUSCO to assess the transcriptome assembly by measuring the
   completeness of the transcriptome based on evolutionary present
   universal single-copy orthologs. The number of transcripts, mean
   length, N50 length, BUSCO scores and percentage of the alignment with
   the pre-processed reads were given in [112]Table S1.

   The number of up- and down-regulated genes, along with the total number
   of genes obtained in each sample comparison, are listed in [113]Table
   1.

   We used RSEM for the quantification of the genes. The count matrix
   generated we then taken as the input by edgeR. The count matrix along
   with the expected count, FPKM and TPM are given in [114]Table S3. The
   downstream analysis resulted in the identification of a total of 14,330
   genes in which 4755 were up-regulated and 9575 were down-regulated. For
   each of the analysis, the total number of genes ranged from 1087 to
   3882. The number of genes up-regulated was in a range of 247 to 1409
   and down-regulated genes were spans from 435 to 3170. Subsequently, the
   assembled transcripts were annotated by BLASTX against a non-redundant
   (NR) protein database, PFAM, KEGG, Uniprot/Swissprot, InterPro and
   STRING databases. It is likely that the cv. Bianca faces a limitation
   of resources around the third week of June, when the first sampling of
   the plant material was made and, that in a month, it reaches its
   maximum peak, corresponding to the second sampling period when the drop
   of inflorescence buds started. June “OFF” vs. July “ON” corresponds to
   the most divergent scenarios. The Venn diagram shows the overlap of the
   genes for “ON” and “OFF” seasons of June and July ([115]Figure S2). The
   figure shows 37,453 genes were common among all the seasons which might
   have a role in the developmental process rather than the alternate
   bearing. The main comparison is focused on “ON” and “OFF” period of
   June to investigate thoroughly different pathways and processes of the
   bud abscission. This comparison can also avoid factors like
   physiological and developmental changes that might occur in the bud
   during the two different time points (June and July). To add strength
   to our conclusion, a comparative study on the effect of crop load
   during the “OFF” and “ON” period of July is also investigated.

3.2. Effect of Crop Load on Photosynthesis During in June “OFF” vs. June “ON”
Inflorescence Buds

   The changes in gene expression in “ON” inflorescence buds do not
   reflect an enhancement of photosynthetic activity when compared to
   “OFF” year inflorescence bud from non-fruiting shoots). Most of the
   genes involved in photosynthesis were up-regulated during the “OFF”
   year. Both photosystem II PSII polypeptide subunits, MAF1, a global
   repressor of RNA polymerase III (Pol III) and PDE335 (Pigment defective
   335) showed an up-regulation during June “OFF” ([116]Table S4). In
   contrast, a gene calling for CRR3 (chloro-respiratory reduction 3) was
   repressed during the “OFF” period. The genes encoding for cytochrome
   (UGT76D1), ATP synthase (PDE332) and cyclic electron flow (PGR5-LIKE A)
   were enhanced during the June “OFF” period. ([117]Table S4).

3.3. Effect of Crop Load on Starch Metabolism in—June “OFF” vs. June “ON”
Inflorescence Buds

   Crop load had much greater effects in reducing starch contents and
   limiting the starch accumulation. Therefore, the study on the
   relationship between crop load and starch metabolism helps in assessing
   the functional distribution of starch in “ON” and “OFF” flower buds.
   The genes encoded for sucrose phosphate synthase (SPS1F), less adhesive
   pollen 5 and starch synthase 4 were enhanced in the tissue of
   inflorescence buds of the “OFF” current year shoots ([118]Figure 2).
   These enzymes are involved in the formation of carbohydrate reserves
   [[119]29]. The genes encoded ALPHA-AMYLASE 3, BETA-AMYLASE 7,
   fructosidase 4, glycosyl hydrolases family 32 and pfkB like
   carbohydrate kinase, which was involved in carbohydrate degradation was
   repressed ([120]Figure 2).

Figure 2.

   [121]Figure 2
   [122]Open in a new tab

   Figure shows the Mapman pathways in sucrose-starch metabolism. Figure
   highlights differentially expressed genes between inflorescence buds in
   the non-bearing shoot (June “OFF”) and inflorescence buds in bearing
   shoot (June “ON”) in sucrose degradation (A) and starch synthesis (B)
   pathways. Individual genes were represented by small squares. The color
   scale indicates the log2 FC value. Red represents up-regulation and
   blue represents down-regulation in June “OFF” relative to June “ON”.

3.4. Effect of Crop Load Status on Polyamine and Transcription Factors in
Inflorescence Buds June “OFF” vs. June “ON” Shoots

   This section of study was conducted to examine the role of free
   polyamines in the inflorescence bud abscission. The “OFF” inflorescence
   buds exhibited significantly higher polyamine (PA) and spermidine (Spd)
   enhancement than the “ON” ones, during most of the period ([123]Figure
   S3). In “OFF” inflorescence buds, the genes encoding for thermospermine
   synthases (ACL5), probable polyamine transporter, Polyamine oxidase 1
   isoform 1 and spermidine synthase (speE) were enhanced. On the
   contrary, the expression of S-adenosyl-L-methionine-dependent
   methyltransferases was repressed.

   In June “OFF” inflorescence buds, three bZIP (bZIP61, leucine zipper
   transcription factor 16 and G-box binding factor 3), ARF7 (Auxin
   response factor 7), WRKY19, zinc ion binding and four homeobox genes
   (Enhanced drought tolerance 1, BEL1-like homeodomain 3, IFL1 and HB-8)
   were down-regulated, while one histone (ULI3) gene, two alfin-like
   members, two MYB factor (MYB103 and MYB14), three WRKY (WRKY31, WRKY72,
   and WRKY53), all the histone related factors and AS2 were up-regulated
   ([124]Table S5). In June “ON” buds, the study reported the enhancement
   of MYB factors (MYB60, MYB3 and MYB106), WRKY factors (WRKY19 and
   WRKY49) and Histone acetyltransferases.

3.5. Gene Set and Pathway Enrichment Analysis During in June “OFF” vs. June
“ON” Inflorescence Buds

   DAVID software was used to identify the biological processes, cellular
   components and molecular functions affected by crop load at
   transcriptomic level considering the differentially expressed genes in
   the June “OFF” and June “ON”. While comparing, June “OFF” buds and June
   “ON” buds, 53 GO terms were down-regulated, whereas 31 were
   up-regulated ([125]Figure S4). The biological pathways that are known
   to be repressed during “OFF” response to salicylic acid, chloroplast
   envelope, circadian rhythm, response to auxin, ion transmembrane
   transport, apoplast and proteolysis were found in our analysis. In
   contrast, we identified some GO-terms that were up-regulated in
   response to alternate bearing, such as nutrients ion transport, ABA
   catabolic process, gibberellin catabolic process, amino acid
   transmembrane transport and carbohydrate metabolic process ([126]Figure
   S4).

3.6. Effect of Crop Load on Hormone Metabolism in June “OFF” vs. June “ON”
Inflorescence Buds

   The objective of the current section was to study the role of hormone
   in inflorescence bud abscission. The genes involved in hormone-related
   categories are summarized in [127]Figure 3.

Figure 3.

   [128]Figure 3
   [129]Open in a new tab

   Figure shows hormone metabolism in Pistachio among the June “OFF” vs.
   June “ON” inflorescence bud comparison. The color scale indicates the
   log2 FC value. Red represents up-regulation and blue represents
   down-regulation in June “OFF” buds relative to June “ON” buds.

   Repression of ethylene and gibberellin pathways were identified in
   inflorescence buds of “OFF”, whereas ABA and IAA pathways were mostly
   up-regulated. In June “OFF” inflorescence buds, five genes responsive
   to ethylene, two genes responsive to gibberellin and two genes
   responsive to cytokinin were down-regulated ([130]Figure 3). Relating
   to auxin-responsive genes, down-regulation of PIN formed 1 and
   up-regulation of TOR, Potassium channel beta subunit 1 (KAB1) and
   Jasmonate resistant 1 were observed. Relating to ABA there was an
   up-regulation in abscisic acid insensitive 3, lipid transfer protein 3,
   shaker potassium ion channel, SNF1, potassium transport 3,
   phosphotransmitter 4 and Carotenoid cleavage dioxygenase 1. Several
   genes involved in ethylene biosynthesis and signaling such as
   Oxoglutarate (2OG) and Fe (II)-dependent oxygenase, Root phototropism
   2, Flavanone 3-hydroxylase, Ethylene response 2 and phosphate
   deficiency root hair defective 1 were repressed during in “OFF” buds
   ([131]Figure 3).

3.7. Effect of Crop Load on Ubiquitin and Autophagy Dependent Degradation in
June “OFF” vs. June “ON” Inflorescence Buds

   The results on the effect of crop load on ubiquitin and
   autophagy-dependent degradation could be a tool for understanding the
   premature inflorescence bud abscission presumably associated to the
   alternate bearing mechanism of P. vera. The genes in inflorescence buds
   from “ON” and “OFF” shoots, that were involved in ubiquitin and
   autophagy-dependent categories were summarized in [132]Figure 4.

Figure 4.

   [133]Figure 4
   [134]Open in a new tab

   Figure shows Ubiquitin and Autophagy dependent degradation in Pistachio
   among the June “OFF” vs. June “ON” inflorescence bud comparison. The
   color scale indicates the log2 FC value. Red squares represent
   up-regulation and blue squares represent down-regulation in June “OFF”
   relative to June “ON”.

   It is worthy to mention that most of the genes were repressed in “OFF”
   inflorescence buds. During this season, genes responsive to autophagy
   (ATG8C: Autophagy related protein 3) and genes responsive to ubiquitin
   proteasome (PAG1: Proteasome Alpha Subunit G1 and Cytochrome P450) were
   down-regulated. While discussing the E3 RING/U-BOX genes, it is worthy
   to mention the down-regulationof PEROXIN 2, BRUTUS (BTS), Malate
   dehydrogenase, Sugar-insensitive 3 and MAPK in OFF buds. Relating to
   the up-regulated genes, we noticed genes such as reactive intermediate
   deaminase A, ARM repeat superfamily protein, B-box domain protein 27,
   ARF23, SKP1 interacting partner 6 and PHY rapidly regulated 2
   ([135]Figure 4).

3.8. Effect of Crop Load on Carbohydrate Metabolism and Mobilization in June
“OFF” vs. June “ON” Inflorescence Buds

   The objective of the current section was to verify the role of CHO
   reserves and mobilization as a cause or effect of the drop of
   inflorescence buds in P. vera. The relationship among the carbohydrate
   metabolism and mobilization pathway in Pistachio and the inflorescence
   buds from non-bearing shoots (June “OFF”) and bearing shoots, June “ON”
   is indicated in [136]Figure 5.

Figure 5.

   [137]Figure 5
   [138]Open in a new tab

   Figure shows carbohydrate metabolism and mobilization pathway in
   Pistachio among the June “OFF” vs June “ON” inflorescence bud
   comparison. The red circle represents the value of log2 fold change.
   The line indicates the effects on carbohydrate levels driven by
   differential expression of different CHO metabolism genes.

   The study showed that the pistachio inflorescence bud of non-fruiting
   shoots “OFF” required low amounts of carbohydrates due to the lack of
   fruits at the time and thus accumulated some starch. Similarly, we also
   found that the inflorescence buds of bearing and non-bearing pistachio
   shoots differed in their carbohydrate storage and mobilization
   patterns, suggesting that the in-season carbon mobilization might
   influence the flower bud abscission directly or indirectly linked to
   the alternate bearing. Raffinose synthase gene (Raffinose synthase 5
   (RS5), two galactinol synthase genes (Galactinol synthase 1 and
   Galactinol synthase 2) and MIOX2 showed repression in the “OFF” buds,
   whereas sugar alcohols, such as callose synthase and
   trehalose-6-phosphate synthase 11, showed an up-regulation ([139]Figure
   5).

3.9. Comparison between “ON” and “OFF” Inflorescence Buds of “JUNE” and
“JULY”

   A comparative study of differently regulated genes among the “ON” and
   “OFF” inflorescence buds collected in “June” and “July” summarizes
   that, during the “OFF” season of July, there is a gradual reduction of
   raffinose synthase 1 and MIOX2 as we identified in June “OFF”. The
   enhancement of hormones like ABA and, at the same time, reduction of
   gibberellin and ethylene indicates that July “OFF” is gradually showing
   the same pattern as that of June “OFF” ([140]Figure S5). SnRK1 and TOR
   down-regulated both the cases; therefore, no programmed cell death
   (PCD) and autophagy occurs during July “OFF” and makes plant stable for
   the next upcoming “ON” season. During the comparison of the
   inflorescence buds in fruiting shoots “ON” of June and July, we could
   find that almost all the genes during July “ON” participate in a
   similar way as that of “ON” June. This comparison proves that gene
   expression profiling associated with “ON” season of June and July and
   “OFF” season of June and July are similar proving the importance of
   these genes in the flower bud abscission and alternate bearing
   ([141]Figure S5).

Effects of Crop Load in July “OFF” vs. July “ON” Inflorescence Buds

   An enhancement ABA was identified in inflorescence buds of July “OFF”,
   whereas ethylene and gibberellin pathways were mostly down-regulated.
   In July “OFF” inflorescence buds, three genes responsive to ethylene
   (ETR2, 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase, Flavanone
   3-hydroxylase) one gene responsive to gibberellin (gibberellin
   2-oxidase 4) and one gene responsive to cytokinin (Isopentenyl
   transferase 5) were down-regulated ([142]Figure S6). Relating to
   auxin-responsive genes, down-regulation of PIN formed 1 and auxin F-box
   protein 5 and the up-regulation of TOR, Potassium channel beta subunit
   1 (KAB1) and Glycoside Hydrolase Family 3 were observed. Relating to
   ABA there was an up-regulation in SNF1, Abscisic acid insensitive 3 and
   Carotenoid cleavage dioxygenase 1 ([143]Figure S6).

   The comparative study of the July “OFF” vs July “ON” produced similar
   results to the results of June “OFF” vs June “ON”. The genes encoded
   for sucrose phosphate synthase (SPS1F) and starch synthase 4 were
   enhanced in the inflorescence buds of the July “OFF”. The genes encoded
   ALPHA-AMYLASE 3, BETA-AMYLASE 7 and fructosidase 4 were repressed
   ([144]Figure S7).

   Most of the genes involved in photosynthesis were up-regulated during
   the July “OFF” year, similar to the results of June “OFF”. The
   photosystem II PSII polypeptide subunits and PDE335 (Pigment defective
   335) showed an up-regulation, whereas gene calling for CRR3
   (chloro-respiratory reduction 3) was repressed during July “OFF”
   ([145]Table S6). The genes encoding for cytochrome (UGT76D1), ATP
   synthase (PDE332) and cyclic electron flow (PGR5-LIKE A) were enhanced
   during the “OFF” period ([146]Table S6).

4. Discussion

   The growth of the endocarp of the cultivar Bianca is from the first
   week of May to the end of June, while the growth of the embryo is from
   the first week of July to the end of August [[147]1]. In “ON” trees,
   most of the inflorescence bud’s abscission starts at the end of June
   and continues in July and August. None of the works to date could
   confirm the involvement of flowering promoter and repressor genes in
   regulating inflorescence bud’s abscission in pistachio. This study
   provides insights into the transcript changes between inflorescence
   buds in bearing and non-bearing shoots in order to identify the
   molecular mechanism causing premature inflorescence bud abscission,
   which is linked to the alternate bearing in the Italian pistachio
   cultivar “Bianca”.

   The relationships between the flower bud drops linked to alternate
   bearing and the carbohydrate storage have been mentioned in several
   studies [[148]2,[149]5,[150]13,[151]30]. It generally seems evident
   that in pistachio trees, nutrients are stored during the “OFF” year and
   that they are used for reproductive growth in the following year
   [[152]2,[153]13,[154]14,[155]31]. There are significant changes in
   starch content and different translocation of starch in the tissues of
   “ON” and “OFF” trees [[156]14,[157]32] and it has been suggested that
   the mobilization of stored carbohydrates may cause inflorescence bud
   abscission in pistachio [[158]8]. The role of individual sugars in the
   process of inflorescence bud abscission has not yet been investigated
   [[159]2].

   In our study, genes encoding for sucrose phosphate synthase (SPS1F),
   degradation sucrose invertase (A/N-InvE), starch synthase 4, callose
   synthase and trehalose-6-phosphate synthase 11 were enhanced in the
   June “OFF” inflorescence buds, whereas BETA-AMYLASE 7, ALPHA-AMYLASE 3,
   branching enzyme 3, fructosidase 4, glycosyl hydrolases family 32, and
   pfkB like carbohydrate kinase were repressed. This supports the
   nutritional theory demonstrating that nutrients are stored during the
   “OFF” year to be used for reproductive growth the following year in
   pistachio trees. Interestingly, in rice and sugar, hormone signals
   regulated ALPHA-AMYLASE 3 enzyme expression, which catalyzed starch
   degradation [[160]33]. In particular, sugar starvation promoted the
   expression of ALPHA-AMYLASE 3 that resulted in the up-regulation in
   pistachio “ON” inflorescence buds.

   Raffinose synthase gene (Raffinose synthase 5 (RS5), two galactinol
   synthase genes (Galactinol synthase 1 and Galactinol synthase 2) and
   MIOX2 showed enhancement in our study in inflorescence buds of bearing
   shoots (“ON”). The raffinose family of oligosaccharides has a wide
   range of predicted functions and are currently emerging as crucial
   molecules during stress response in plants [[161]34], because of their
   membrane-stabilizing, antioxidant and, perhaps, predictable signaling
   functions [[162]35]. They participate in several cellular functions,
   such as transport and storage of sugars [[163]36], signaling molecule
   following pathogen attack and wounding [[164]37], signal transduction
   [[165]38], membrane trafficking [[166]39] and mRNA export [[167]40].
   Recent transcriptional profiling data in Arabidopsis thaliana showed
   up-regulation of the Myo-inositol oxygenase (MIOX) genes under limited
   energy or nutrient conditions shows consistent with our results
   indicating an up-regulation in “ON” buds. MIOX2 plays a prominent role
   in the oxidation of inositol for the needs of the plant in different
   tissues and it is involved in the biosynthesis of nucleotide sugar
   precursors for cell-wall matrix polysaccharides [[168]41].

   Interestingly, trehalose-6-phosphate (T6P), that was up-regulated in
   “OFF” inflorescence buds, seem to play a central role in sugar
   metabolism regulation in plants [[169]42]. It has been proposed that
   T6P is transported by an unknown mechanism into plastids, where it
   induces starch synthesis via thioredoxin-mediated activation of AGPase,
   and that there is a regulatory loop which involves T6P, SnRK1 (a gene
   that represses plant growth, inhibited by T6P) and bZIP11 that control
   sucrose availability and utilization. In source leaves, T6P fine-tunes
   sucrose levels by adjusting sucrose synthesis, while it regulates
   Sucrose consumption in sink organs, probably acting via multiple
   mechanisms, including inhibition of the SnRK1 gene [[170]43]. T6P
   regulates growth in relation to sucrose supply by adjusting
   biosynthetic reactions and through regulating hormone signaling like
   auxin either directly or indirectly [[171]44].

   In the pistachio tree, it has been demonstrated that the accumulation
   of nitrogen, phosphorus and potassium is greater in inflorescence buds
   of non-bearing branches, compared to the ones of the bearing branches
   [[172]10], and that the nutrient contents of the trees and annual
   nutrient consumption are influenced by the alternate bearing [[173]10].
   Our results showed an enhancement of nitrogen permease regulator of
   amino acid transport activity 3 and carbon-nitrogen hydrolase, which is
   supporting the fact that the concentration of nitrogen (N) was higher
   in the inflorescence buds, leaves, and fruits of non-fruiting branches
   (OFF) than in the analogous “ON” structures [[174]10]. Competition
   between flower buds and developing nuts for N might play an important
   role.

   Our study showed up-regulation of potassium ion channel, magnesium
   dechelatase (SGR), magnesium-chelatase subunit (ChlH), CSC1-like
   protein (Calcium-dependent channel) and calcium permeable stress-gated
   cation channel (TMEM63) during in “OFF” inflorescence buds. Some
   studies found that N, K, Ca and Mg content were affected by crop load
   in olive leaves, showing lower values following the “ON” year
   [[175]45]. However, the information on the effects of fruiting on
   nutrient concentrations of different organs of pistachio trees relative
   to bud abscission is limited.

   In the “OFF” inflorescence buds of June, we found genes encoding for
   sugar phosphates accumulation including substrates of the Calvin cycle,
   glycolysis, and the pentose phosphate pathway. Sugar phosphates
   transformed into sucrose and transport to fruit. This can reduce the
   sugar phosphates in the source tissues of trees with strong sink tissue
   such as fruit. Whereas, in the inflorescence buds of “OFF” shoots, the
   absences of fruits lead to the accumulation of sugar phosphates and
   starch. Studies showed that the expression of some of the genes and
   proteins involved in the Calvin cycle is up-regulated in “OFF” trees
   [[176]18].

   Studies on the stomatal transpiration rates in another alternating
   species, such as the olive tree, have not shown any variation between
   plants in “ON” and “OFF” [[177]46], contrary to what occurs in species
   like orange or strawberry [[178]47]. Studies on photosynthesis and
   production of photosassimilate in pistachio have shown a decline in
   “ON” trees during mid-July, which could be due to early senescence and
   the fall of the leaves [[179]48]. A similar decline in photosynthesis
   due to leaf aging has been reported for apple trees [[180]49] and olive
   trees [[181]46]. In the present study, the up-regulation of both the
   PSII polypeptide subunits of the photosystem II, MAF1, a global RNA
   polymerase III (Pol III) and PDE335 (defective Pigment 335) repressor
   that can be found in the “OFF” buds of June indicate that pistachios
   have the ability to maintain relatively high photosynthetic rates.

   In plants, nutrient limitation due to sink competition leading to sugar
   starvation is perceived as nutritional stress and generate changes in
   the redox status promote the synthesis of free radicals which can cause
   transient oxidative stress due to an increase of ROS generation
   [[182]50], that can be neutralized by some adaptive mechanisms which
   can protect the cells from oxidative damage. The cells subjected to
   sugar starvation at the beginning try to adapt to this deficiency
   through a gradual metabolic reorganization that implies the
   substitution of carbohydrate metabolism by protein and lipid metabolism
   and that change may cause autophagy [[183]51]. Variations in sugar
   levels induce changes in ROS production and sugar starvation can cause
   the activation of ROS production, as indicated by transcriptome
   profiling analysis, where sucrose starvation results in activation of
   oxidative stress genes, such as catalase [[184]52].

   It has been found that plant processes, such as cell division,
   morphogenesis and stress responses, were affected by the involvement of
   polyamines (PAs)-putrescine (Put), spermidine (Spd), spermine (Spm),
   cadaverine (Cad) and thermospermine (t-Spm) [[185]53]. The free
   polyamines could have an important physiological function in the
   development of flower bud abscission, which causes alternate bearing in
   pistachio trees [[186]13]. A significant decrease in polyamines (Pas;
   Put and Spd) in shoots and leaves of “ON” trees during the heavy bud,
   abscission period was reported while an increase detected during the
   same period in “OFF” trees, indicating an association between flower
   bud abscission and the level of PAs in pistachio. In Satsuma mandarin,
   during the “ON” season, polyamines were accumulated in the stem which
   can suppress flowering and cause fruit bearing [[187]54]. It is
   possible that a decrease in N concentrations in plant tissues may cause
   a decrease in polyamines, as they can represent nitrogenous sources or
   as signal molecules that regulate the fruitlet abscission process in
   grapevine [[188]55]. Many studies have highlighted that abscission or
   ethylene biosynthesis can be delayed with low levels of
   S-adenosylmethionine (SAM). During this phase, PAs and ethylene compete
   and PAs can become dominant. The low concentrations of PAs can trigger
   the senescence and cause abscission [[189]56].

   In our study, polyamines related genes exhibited significantly higher
   enhancement in the inflorescence buds of non-fruiting branches (OFF)
   than the “ON” fruiting ones, in accord with the recent study of
   Gündeşlí [[190]15]. The genes encoding for thermospermine synthases
   (ACL5), probable polyamine transporter, Polyamine oxidase 1 isoform 1
   and spermidine synthase (speE) were enhanced during the “OFF” seasons.
   These references, along with our results, support the fact that