Abstract

   Proceeding to illumina sequencing, determining RNA integrity numbers
   for poly RNA were separated from each of the four developmental stages
   of cv. Summer Black leaves by using Illumina HiSeq^™ 2000. The sums of
   272,941,656 reads were generated from vitis vinifera leaf at four
   different developmental stages, with more than 27 billion nucleotides
   of the sequence data. At each growth stage, RNA samples were indexed
   through unique nucleic acid identifiers and sequenced. KEGG annotation
   results depicted that the highest number of transcripts in 2,963
   (2Avs4A) followed by 1Avs4A (2,920), and 3Avs4A (2,294) out of 15,614
   (71%) transcripts were recorded. In comparison, a total of 1,532
   transcripts were annotated in GOs, including Cellular component, with
   the highest number in “Cell part” 251 out of 353 transcripts (71.1%),
   followed by intracellular organelle 163 out of 353 transcripts (46.2%),
   while in molecular function and metabolic process 375 out of 525
   (71.4%) transcripts, multicellular organism process 40 out of 525
   (7.6%) transcripts in biological process were most common in 1Avs2A.
   While in case of 1Avs3A, cell part 476 out of 662 transcripts (71.9%),
   and membrane-bounded organelle 263 out of 662 transcripts (39.7%) were
   recorded in Cellular component. In the grapevine transcriptome, during
   the initial stages of leaf development 1Avs2A showed single transcript
   was down-regulated and none of them were up-regulated. While in
   comparison of 1A to 3A showed one up-regulated (photosystem II reaction
   center protein C) and one down regulated (conserved gene of unknown
   function) transcripts, during the hormone regulating pathway namely
   SAUR-like auxin-responsive protein family having 2 up-regulated and 7
   down-regulated transcripts, phytochrome-associated protein showed 1
   up-regulated and 9 down-regulated transcripts, whereas genes associated
   with the Leucine-rich repeat protein kinase family protein showed 7
   up-regulated and 1 down-regulated transcript, meanwhile Auxin Resistant
   2 has single up-regulated transcript in second developmental stage,
   although 3 were down-regulated at lateral growth stages (3A and 4A). In
   the present study, 489 secondary metabolic pathways related genes were
   identified during leaf growth, which mainly includes alkaloid (40),
   anthocyanins (21), Diterpenoid (144), Monoterpenoid (90) and Flavonoids
   (93). Quantitative real-time PCR was applied to validate 10
   differentially expressed transcripts patterns from flower, leaf and
   fruit metabolic pathways at different growth stages.

Background

   Vitaceae comprised of 14 genera and about 900 species which are
   distributed all over the globe particularly in tropical regions of
   Australia, Asia, Africa and the Pacific Islands with a small number of
   genera in temperate areas [[40]1]. Vitis is one of the economically
   most important and globally cultivated fruit crop, covering about 8
   million hectares of area and producing about 67.5 million tons
   ([41]http://www.oiv.int/) of grape berries, with highly valued products
   such as juices, liquors and wines [[42]2].

   Leaf is the most important vegetative organ supplying the energy,
   nutrition and hormones for the fruit growth and development. Thorough
   knowledge of the ongoing biological networks during the leaf growth is
   very important. So leaf growth and development in all vascular plants
   is begun with the growth of meristem tissues. In plants, subsequent
   cell multiplication and elongation occurs primarily from a basal
   intercalary meristem producing a gradient of cells along the leaf, with
   the sequence of interconnected and overlapping phases: initiation,
   general cell division, transition, cell expansion and meristemoid
   division phases [[43]3]. Consecutive transverse segments of leaf
   therefore provide harmonized large number of cells at various
   developmental stages. Successive transverse sections of a leaf provides
   synchronized number of cells at different growth stages [[44]4]. The
   hypotheses of stage specific genes are more important for growth is
   also support the findings of Fasoil et al., [[45]5], he reported that
   the organ identity in the grapevine transcriptome is less important
   than the developmental stage. Few organ-specific genes were shared
   among the different developmental stages; however up to 16% of the
   organ-specific genes expressed in the flower were common to the
   different floral organs.

   Photosynthetic differentiation is essential for vascular plants;
   however it is not clearly understood. Although various pathways related
   to leaf development have been thoroughly reviewed, to understand the
   viewpoint of the single cell has not been discovered scientifically
   [[46]6]. The studies were focused mainly on leaf development and
   networks of genes signaling, that are linked with the conscription of
   cells from meristem develops into leaf primordia, the organization of
   abaxial-adaxial polarity and the expansion of the blade laminal
   [[47]7]. As a result, an incomplete thoughtful knowledge about
   signaling coordination that compels photosynthetic progress from
   proplastid to chloroplast [[48]8]. Leaf development is concerned with
   the photosynthetic productivity which underlies the global challenges
   such as climate change, bio-energy and ensure food security; provides
   thoughtful knowledge and enables us to operate photosynthetic
   activities [[49]3]. Additional research work predicted the preliminary
   knowledge about the mechanisms that direct the trafficking of
   chlorophyll metabolic intermediates in leaves. In plants photosynthetic
   activities are more complex due to chloroplastic and cellular
   dimorphism. photosynthesis relies on Kranz-type leaf anatomy in which
   the veins running the size of the leaf are enclosed by two layers
   [[50]9]. While much progress has been made in defining growth
   regulators signaling and biosynthetic pathways, but the regulation by
   ecological and growth signals remain not well reported. De novo auxin
   biosynthesis plays an indispensable role in plant growth. The growth
   hormone auxin, which is mainly symbolized by indole-3-acetic acid, is
   concerned with the regulation of plant development. Even though IAA was
   the primary plant hormone acknowledged, the biosynthetic pathway at the
   genetic level has remain unclear [[51]10].

   Transcriptome sequencing using NGS technologies have been increasingly
   carried out in model as well as in non-model plants for gene detection
   and advancement in markers development [[52]11, [53]12]. Due to the
   rapidly developing technology of NGS, the quantity of sequencing data
   that could be produced in experiment has radically increased in current
   years, as the total length of the sequencing reads. This has led to a
   better level of transcriptome exposure, enhanced the specificity and
   precision as in mapping sequencing reads. Significantly, constant
   incremental developments in defining the grapevine transcriptome in the
   form of functional annotation [[54]13, [55]14] and gene ontology
   assignment [[56]15], now permits the precise narration of the
   functional task of mainly about Vitis vinifera genes [[57]16].
   Particularly analogous RNA deep-sequencing presents modern scientific
   platform for scrutinizing transcriptional instruction. It makes
   possible the accurate elucidation of transcripts present surrounded by
   a specific sample, and could be applicable to work out gene function
   based on unconditional transcript abundance [[58]17]. In the single
   acknowledgment, in grape vine Zenoni, Ferrarini [[59]18] produced RNA