Modified plant structures are termed as galls that are induced by other organisms such as bacteria, viruses, and insects. Gall forming insects often obtain easy food access and gain protection from harsh environmental conditions and natural enemies. Gall formation is a highly adaptive and evolutionary advantageous phenomenon (9). Pistacia from the Anacardiaceae family known for its leaf galls induced by Baizongia pistaciae L are of ethno medicinal importance. Pistacia gall has the ability to resolve several health issues like asthma, dysentery, psoriasis, snakebite etc (10). A very little is known about the mechanism of gall formation and the way insect and plant recruit plant biochemical, physiological and developmental pathways for their own benefit.
Next Generation Sequencing technology was applied to sequence non-model species such as Pistacia. The estimated genome size of Pistacia is ~ 549 MB based on whole genome assembly. The SSR (di, tri, and tetra) predicted from Pistacia genome. SSRs are useful tools to identify genetic hotspot and diversity in natural populations, gene bank. These markers will be useful in fingerprinting and authentication of Pistacia Ayurvedic formulations. They can be used for genetic linkage mapping and evolutionary studies and have a high level of transferability between closely related species (11).
From the transcriptome analysis, we found that organic substrate metabolic process (14338 transcripts) representing biological process was higher in gall compared to leaf (5932 transcripts). Similarly intrinsic component of membrane (8096 transcripts) was higher in gall representing cellular component. Higher organic cyclic binding (11367 transcripts) were higher molecular functions in gall when compared to leaf (4982 transcripts). We also found an abundance of enzyme types in gall when compared to leaf. As enzymes are associated with various biological processes, the identification of important enzyme codes might provide us information on important functional pathways and metabolic activities between gall and leaf tissue of Pistacia (12, 13). These results suggest that higher metabolic function and enzyme activity in gall when compared to leaf.
In our studies we found that genes coding for terpene synthesis such as beta-amyrin 28-oxidase (72%), hydroxymethylglutaryl-CoA synthase, Farnesyl pyrophosphate synthase, beta-amyrin synthase 1-like, gamma-terpinene synthase and squalene synthase were highly expressed in gall tissue as compared to leaf. Among these, terpene synthesis genes such as geranylgeranyl pyrophosphate synthase family protein and geranyl diphosphate synthase are highly expressed in gall compared to leaf. It was reported that Baizgonia pistaciae - induced galls showed high accumulation of monoterpenes levels (14).
Terpenes being the most structurally diverse class of plant metabolites play a vital role as pollinator attractants, growth regulators, and stabilizers of membrane structure as well as in mediating direct and indirect plant defences. The key enzymes responsible for monoterpene formation in plants are monoterpene synthases. In the previous studies it has been reported that total monoterpene synthase activity is tenfold higher in galls as compared to leaves. The biosynthesis machinery has helped aphids to manipulate the enzymatic system of their host plant and recruit for their own benefit (9).
We observed ethylene responsive, GATA, bHLH, MYB, BZIP, Trihelix, MADS, B3 domain, nitrogen assimilation, WRKY, steA, UNE12, NAC, MIZ, AS1, C6, fungal specific domain and GRAS transcription factor families identified in the gall tissue. Studies WHO have been reported that transcription factors play an important role in regulation of metabolic process. MYB proteins are associated with regulation of secondary metabolism, control of cellular morphogenesis and regulation of meristem formation and the cell cycle (15). The bHLH (basic helix-loop-helix) family have been reported in controlling cell proliferation and development of specific cell lineage (16).
We have also found major facilitator superfamily (MFS) transcripts, multidrug transporter and ABC transporter permease. Environmental stresses are the major factors that contribute to plant growth and development. It has been found that many transporter families of plants participate in responding to pesticides, toxic chemicals and infection of pathogens. The transporters from plants are classified into five families such as ATP binding cassette (ABC), multidrug and toxic compound exporters (MATE), small multidrug resistance (SMR), resistance-nodulation-division proteins (RND), and major facilitator superfamily (MFS). Multidrug transporters are the group of membrane proteins in MFS known to have a role in the regulatory network of responding to the stress by which they act as drug/H + transporters to reduce the accumulation of toxin in the body (17).
In this study we also observed that GA receptor and scarecrow-like protein 8 - DELLA proteins have highly expressed in gall. Wang et al 2016., have reported that exogenous Gibberellic Acid (GA) application promote gall development. Giberellin receptors and DELLA proteins have been reported to have major role in signal transduction of GA in plant development (8).
In the current study identified serine/threonine-protein kinase transcripts highly expressed in the gall tissue. Gall development through aphid infestation on Pistacia apical meristem is complex physiological process which involves active attack by aphids, passive defence of plant, confrontation between the host and the aphid, and mutual coordination between the two. Therefore it is expected that transcripts linked to plant-aphid interaction would be enriched in the gall tissue. It has been reported WHO that protein phosphorylation is one of the most important events in the disease resistance pathway. Serine/threonine-protein kinases in plant cells are referred to ‘central processor unit’, as they receive information from receptors in terms of environmental stress, phytohormones, and other external factors, and bring a changes in metabolism, gene expression, and cell growth and division (18).
Pistacia gall has been used in Ayurveda formulation. White cottony layer present inside the gall may be the insect excretion and might also be the possible metabolites synthesised from plant-insect interaction. In order to identify whether any insect proteins were present inside the gall, the white cottony layer inside gall was analysed for proteins. We also found that one of the transcripts in RNA seq data was also mapped to tubulin of pea aphid (GCA_000142985.2Acyr_2.0). Tubulin is known to express differently at different stages of development in insects. Tubulins are the building blocks of microtubules and these tubulins have major role in maintaining cell shape, movement and intracellular transport of substances. Tubulins are the essential components of cytoskeleton and spindles (19). Therefore, the study on tubulin and its information helps to understand the expression analysis of the other genes in Pistacia. We hypothesize that the actin filaments and microtubules are secreted from the insects and deposited in the galls. In the future, it would be interesting to profile metabolites from the gall and elucidate the molecular interactions. This study provides a basis for the understanding of the genes expressed in gall when compared to leaf and also genes required for gall induction and development.