Gene transcripts responsive to drought stress identified in Citrus macrophylla bark tissue transcriptome have a modified response in plants infected by Citrus tristeza virus

Abstract

Citrus macrophylla Wester (CM) has valuable agronomic characteristics such as the ability to grow in saline soils, although with low tolerance to prolonged drought stress (DS). To understand the mechanisms that characterize CM response to water scarcity, this study compared transcriptome profile changes in CM stem tissue when exposed to DS and identified a total of 2745 differentially expressed transcripts (DETs, fold change > 2), of which 631 were up-regulated and 2114 were down-regulated. DETs up-regulated by DS were enriched in pathways such as the redox and osmotic system or soluble carbohydrates and in transcripts for low molecular weight proteins such as late embryogenesis abundant protein (LEA). Down-regulated transcripts were mainly assigned to photosynthesis, transport, phenylpropanoids, calcium dependent kinases, brassinosteroids and other hormones including salicylic acid and abscisic acid. To assess the interplay between DS and Citrus tristeza virus (CTV) infection, twelve genes were profiled by quantitative Real-Time PCR (qPCR) analysis in control and CTV-infected CM plants, with or without DS. The twelve analyzed transcripts were significantly correlated (r = 0.82, p < 0.001) with the RNA-Seq results and gave insight into the responses of CM to drought and/or to infection with CTV. Transcriptome results unveiled highly responsive genes to DS in stem tissue, which may be candidates for genetic selection of high drought tolerant plants of CM.

Introduction

The genus Citrus (family Rutaceae) is one of the most widely cultivated fruit crops worldwide (Liu et al., 2012). The Mediterranean basin is a leading citrus production region (FAO, 2017). Citrus species that are commonly cultivated in the Mediterranean are poorly drought tolerant and insufficient irrigation is a source of stress, that restricts plant growth and crop yield (De Ollas et al., 2019). The foreseen decrease in rainfall and rise in summer temperatures in the near future with increasingly long periods of drought will have devastating consequences for citrus sector profitability and sustainability (De Ollas et al., 2019). The south-western Mediterranean region is expected to be particularly affected by rising temperatures and drought, which will be highly detrimental for the cultivation and production of citrus fruits (De Ollas et al., 2019; Fares et al., 2017). One possible solution to future challenges linked to water shortage is to breed citrus species less sensitive to drought stress (DS). To make this possible, a better understanding of the molecular mechanisms underpinning drought resistance is essential and may contribute with new candidate genes for improvement and adaptation of citrus crops to future climate conditions.

Extensive studies on DS exist for the model plant Arabidopsis thaliana, and this has contributed to identify DS-related pathways in plants (Seki et al., 2002). Such studies revealed two main strategies that plants have to cope with DS, (i) dehydration avoidance and (ii) dehydration tolerance, which involve different physiological processes (Santos et al., 2021). Dehydration avoidance comprises strategies to prevent water loss, such as a higher efficiency of water absorption by the root system, production of osmoprotectant solutes and proteins, stomatal closure, reduction of leaf area, cell wall hardening and detoxification of reactive oxygen species (ROS) (Dutra de Souza et al., 2017; Hussain et al., 2018). Dehydration tolerance protects cells from serious injury when avoidance systems are insufficient and involves morphological, physiological, and biochemical changes.

DS tolerance of citrus species gains importance depending on whether they are rootstocks or not. Citrus rootstocks have recognized effects on tree size, yield and fruit quality due to physiological, morphological and hydraulic conductivity of the root system (Robles et al., 2017) and thus impact on the scion × rootstock response to DS (Barry et al., 2004; Gonçalves et al., 2016; Santana-Vieira et al., 2016). Molecular responses to DS of citrus rootstocks such as Citrus macrophylla (CM) and other citrus genotypes has mainly been studied in roots, as well as in the leaves of grafted scions, as some rootstocks may be able to transfer their survival strategy to the grafted scions (Dutra de Souza et al., 2017; Molassiotis et al., 2016; Ruiz et al., 2018; Santana-Vieira et al., 2016; Zaher-Ara et al., 2016). Studies using complementary DNA (cDNA) - Amplified Fragment Length Polymorphism (AFLP) analysis of leaf samples from two-year-old C. reticulata × C. sinensis (Amakusa tangor), a plant sensitive to water scarcity grafted on trifoliate orange, revealed 255 differentially expressed transcripts (DETs) out of 6245 transcript-derived fragments. While genes related to the regulation of transcription, defense, energy and transport were up-regulated, other ones related to photosynthesis and to the basic metabolism were down-regulated (Xiao et al., 2017). Another study using cDNA microarrays from leaves and roots of two-year-old ‘Clemenules’ (C. clementina hort. ex Tanaka) grafted on Cleopatra mandarin (Citrus reshni hort. ex Tanaka) identified about 6000 DETs related to citrus DS tolerance. These included dehydrin transcripts and other DETs related to lysine catabolism, proline and raffinose synthesis, zeaxanthin synthesis, hydrogen peroxide reduction, vacuolar malate transport, rare cold inducible (RCI2) proteolipids and defense proteins such as osmotin and heat-shock proteins (HSP) (Gimeno et al., 2009). Studies of two-month-old Rangpur lime (Citrus limonia Osbeck) roots, a rootstock with high tolerance to water stress, using Expressed Sequence Tags (ESTs) revealed up-regulation of transcripts for aquaporins, dehydrins, sucrose synthase, protein kinases, cysteine proteinases and proline-related synthases (Boscariol-Camargo et al., 2007). More recently, RNA sequencing (RNA-Seq) from leaves of C. sinensis grafted onto the drought-tolerant Rangpur lime identified 1764 DETs related to cell wall, carbohydrate and antioxidant metabolism, responses dependent on ABA regulation as transcripts encoding transcription factors, protein kinases and protein phosphatases. Down-regulated transcripts were mainly related to starch metabolism, light reactions and ethylene signaling (Gonçalves et al., 2019). Roots of C. sunki (Hayata) hort. ex Tanaka rootstock, both ‘Sunki Maravilha’ and ‘Sunki Tropical’ grafted with C. sinensis, have different DS tolerance mechanisms. Data revealed that the metabolism of ‘Sunki Maravilha’ with tolerance to drought was able to maintain plant survival and included up-regulation of transcripts related to cell wall metabolism, ABA biosynthesis, carbohydrate metabolism and oxidoreductases, as well as those involved in the regulation of plant responses such as transcription factors and protein (Santos et al., 2021). Plants like ‘Sunki Tropical’ with dehydration avoidance mechanisms showed distinct physiological processes related to high efficiency of water use and maintenance of plant growth/productivity, with up-regulation of DETs associated with cell wall hardening to decrease water loss, as well as to ABA, IAA, ethylene, gibberellins and brassinosteroids (BR) synthesis (Santos et al., 2021). The expression of brassinosteroids improves plant response to abiotic stresses by strenghtening photosystem II and the growth of the root system during water scarcity (Dutra de Souza et al., 2017; Santana-Vieira et al., 2016; Santos et al., 2021).

Under drought conditions, water scarcity is perceived by the roots as a stress signal that travels towards the leaves via the xylem to induce the phytohormone signaling pathway and secondary metabolite biosynthesis. DS also involves the dynamic phloem system, as the phloem sap in sieve elements is the likely route of transmission of signaling molecules that can trigger a global response, so the plant adapts and survives (Evert, 2006; Oberhuber et al., 2017). The identification of genes associated with the stress response is a fundamental step toward rapid improvement of drought tolerance in citrus species using biotechnological methods. Particularly since traditional citrus breeding approaches are time-consuming due to their complex reproductive biology, including high heterozygosity, apomixis, polyembryony and cross- and self-incompatibility (Zhang et al., 2018).

CM or alemow have been extensively used as a lemon rootstock in California and in the Mediterranean region since it induces vigour in citrus scions, and promotes early bearing, very high yield and excellent adaptation to calcareous and saline soils (Levy et al., 1993; Romero-Trigueros et al., 2020). CM takes water up efficiently from well irrigated soils but has low water stress tolerance in case of prolonged drought, with a notorious decreased pruning weight (Robles et al., 2017). Understanding the mechanisms that characterize CM responses to water scarcity is essential for the genetic manipulation of this rootstock with the aim of improving its growth under water stress conditions and a good response to a regulated deficit irrigation. In addition to water scarcity, field plants are also exposed to a diversity of biotic factors and CM is considered one of the most susceptible citrus hosts of Citrus tristeza virus (CTV), commonly distributed in citrus regions worldwide (Dawson et al., 2013). CTV is a single-strand RNA(+) virus that belongs to the Closteroviridae family and only infects Citrus spp. and citrus relatives within the Rutaceae (Karasev et al., 1995). Severe isolates of CTV are causal agents of the two major host disease syndromes: stem pitting, that induces abnormal phloem development and quick decline or Tristeza disease, that may induce plant death, syndromes which pose problems for commercial citrus cultivars. Infection with a severe CTV isolate induces alterations in many metabolic pathways and altered levels of metabolites including amino acids, sugars, sugar acids, signaling molecules (Killiny et al., 2017) as well as metabolites with reactive oxygen scavenger action and enzymes related to oxidative metabolism (Silva et al., 2019), causing overlapping effects to drought stress in plants. Nowadays, CM has a limited use as a lemon rootstock due to its high susceptibility to severe CTV strains (Killiny et al., 2017; Ruiz et al., 2018).

To understand the molecular responses of the CM rootstock to DS, bark slip tissues, which do not include the xylem or pith, were evaluated using Illumina RNA-Seq technology, and transcriptome profiles from control and drought-stressed plants were compared. This strategy allowed evaluation of both the DS response translocated in sieve elements between photosynthetically active tissues and sink tissues, as well as in the external layers of the stem (namely in cortical parenchyma, secondary phloem, periderm and epidermis). Quantitative Real-Time PCR (qPCR) assays were performed to evaluate the changes in expression of twelve selected DETs in control vs. DS-subjected CM, combined or not with infection with a CTV stem pitting isolate T318A (Ruiz-Ruiz et al., 2006). This approach permitted investigation of pathways in CM under DS as well as the identification of specific genes that respond to both abiotic (DS) and biotic (CTV) stress factors.