Introduction

Caesalpiniaceae is a large family with several pharmaceutical applications as being mainly used as a laxative and to relieve constipation. Cassia angustifolia, formally Senna angustifolia (2n = 28) is known for its important applications in pharmaceuticals. C. angustifolia is a wild medicinal drought-tolerant shrub (Ayoub 1977, Khalid et al. 2012) with many cathartic properties (Lemli 1986, Folkard 1995, Hammouda et al. 2005). Drought stress-related genes have been studied at the trancriptomic level in senna (C. angustifolia) (Mehta et al. 2017) as well as in many other plant species such as parsley (Li et al. 2014a,b), bean (Hiz et al. 2014), chrysanthemum (Xu et al. 2013), tall fescue (Hu et al. 2014), and grapevine (Rocheta et al. 2014). The lucine-rich repeat kinase family was recently found in senna (C. angustifolia) as the most abundant group of protein kinases under drought stress in addition to several families of transcription factors (e.g., bHLH, and bZIP, etc.) (Mehta et al. 2017). Previous studies on senna (C. angustifolia) deciphered some physiological, morphological and molecular mechanisms allowed the plant to tolerate drought stress (Ratnayaka and Kincaid 2005, Mehta et al. 2017).

Availability of water is a major obstacle for agricultural productivity. Wild plants growing in severe arid climates provide tools for studying plant response to extreme drought conditions. Generally speaking, drought, salt and heat stresses have large impacts on plant growth and productivity. Other abiotic deleterious stresses nowadays include increased chemicals and pollutants. Besides, it is likely that plants are exposed to more than one type of stress at a time. In particular, drought stress is a major threat in at least 26 % of world’s arable land (Blum 1988). The effects of drought include delayed or stunted growth as well as impaired physiological processes such as photosynthesis, respiration, and mineral exchange (Do et al. 2013). Therefore, it is crucial to get a better understanding the molecular and physiological impacts of drought stress in order to find solutions to help the plant cope with or at least lower the influence of this stress for the sake of maintaining crop productivity and possibly cultivate more crops in arid lands to mitigate global food crisis.

Plants are adapted to drought either by avoidance or tolerance, as the two main strategies, by which a crop plant can maintain yield components and minimize the loss due to the stress. Avoidance mechanisms include the occurrence of several morphological changes, such as stomatal closure and reduced leaf area to reduce respiration, as well as enlarging root systems in order to gain more water with the same intensity of cultivation (Levitt 1980, Budak et al. 2013, Rama Reddy et al. 2014). Alternatively, drought tolerance is a subject of intense research as it mainly occurs due to several physiological and molecular mechanisms that help the plant to adapt with the osmotic pressure due to the shortage of water (Bartels and Sunkars 2005). Tolerance mechanisms were proven to be genetically-dependent as different plant species have different strategies to cope with the problem. These strategies are supported by complex metabolic pathways that should link together and cross-talk in order to produce osmolytes and protein chaperons to secure the cell from the stress and avoid denaturation or damage of important compounds in the cell (Yamaguchi-Shinozaki and Shinozaki 2006, Kantar et al. 2011, Shanker et al. 2014). There are several abiotic stress-related enzymes like glutathione reductase, catalase, superoxide dismutase considered as biomarkers for drought stress tolerance (Khammari et al. 2012).

The present study aim at studying drought-related dynamics of leaf transcriptome in senna (C. angustifolia) to detect the crosstalking pathways possibly associated with drought stress tolerance to add to our understanding of the molecular mechanisms underlying drought stress tolerance in wild plants.

Material and Methods

Plant material sampling and watering regime

The field experiment of sample treatment and harvesting was conducted for senna (C. angustifolia) shrubs grown near Jeddah, Saudi Arabia. Three plants of equal size were selected in which leaf samples were harvested in two consecutive days at three time point of the day (1 h post-dawn, midday and 1 h post-dusk). At dawn of the day 2, plants were watered (25 liters dH₂O) and leaf samples were harvest at the same three time points.

RNA-Seq and KEGG Analyses

Total RNA was extracted from three similar-sized (10 mm²) leaf discs per plant of Cassia angustifolia, then shipped to Beijing Genome Institute (BGI), China, for next-generation sequencing. Recovered RNA-Seq datasets were de novo assembled using the Trinity RNA-Seq Assembly package (r2013-02-25) with optimized parameters and K-mer size set to 25 (Zhang et al. 2015). Differential expression and cluster analysis were done by EdgeR (version 3.0.0, R version 2.1.5). All predicted CDS were annotated against protein database in order to assign putative function of the transcriptome after translation into protein. To identify the biological pathways with enzymes that differentiate at midday samples, the detected genes were mapped to reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.ad.jp/kegg/). RNA-Seq datasets of Cassia angustifolia were validated via qRT-PCR (data provided upon request).

Results and Discussion

Preliminary Data Analysis

Sequence assembly resulted in a number of ˃5000 regulated genes due to watering and the number of annotated genes ˃2000 genes. The term enrichment in this study refers to the increase of a given enzyme or metabolite in the second day due to watering, while suppression indicates that the intensity of the enzyme or metabolite was reduced due to watering. In other words, enzyme or metabolite enrichment indicates that the encoding genes were highly expressed after watering, while repression indicates that the expression of the encoding drought-related genes is abolished as it is no longer required after watering. GO classification indicated that the subcategory “response to stimulus” is repressed as the stress in the second day is completely relieved. We expected that several biological processes of this subgroup can confer tolerance to drought stress.

KEGG Analysis

In order to study the enzymes in selected biological pathways of Cassia angustifolia whose genes encoding them are highly (≥5 FC) regulated due to watering, we mapped the detected genes to reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.ad.jp/kegg/). Drought tolerance is a multigenic process with different metabolic pathways affecting plant growth (Mehta et al. 2017). Pathways that were either enriched or repressed due to watering were examined (Table S1). Of which, we selected the pathways that might have emphasis on plant response to abiotic stress (Table 1). In addition, we have investigated few other pathways whose enzymes were suppressed due to watering, while showed no enzymes that were enriched due to the stress. When applying the criteria of ≥5 FC gene regulation in KEGG analysis, a number of five groups of pathways including 14 subgroups with 22 pathways were selected. These groups are “metabolism”, “Genetic Information Processing”, “Environmental Information Processing”, “Cellular Processes” and “Organismal Systems”. Of which, 10 pathways showed no enrichment in their enzymes due to watering, rather they were repressed due to watering. This indicates that these pathways likely participate in the molecular mechanisms utilized by the organism to cope with the stress, whose enrichment is not required when water becomes available. These pathways are “Biosynthesis of siderophore group nonribosomal peptides”, “Spliceosome”, “VEGF signaling pathway”, “Jak-STAT signaling pathway”, “Autophagy – other”, “Tight junction”, “Longevity regulating pathway”, “Longevity regulating pathway – multiple species”, “Circadian rhythm” and “Thermogenesis” (Table 1). Enzymes or metabolites of the enriched and suppressed pathways are shown in Tables 2 and 3, respectively. The number of activated enzymes due to watering was 43 enzymes, while the number was 107 for the suppressed enzymes due to watering (Tables 2 and 3).

Table 1: Selected pathways of C. angustifolia with enzymes encoded by genes highly (≥5 FC) regulated due to the watering. Green box = pathway enriched due to watering, red box = pathway suppressed due to watering, blue box = regulated TOR-dependent pathway.

Table 2: Selected pathways of C. angustifolia with enzymes encoded by genes highly (≥5 FC) enriched at midday due to the watering. N = before watering, NR = after watering.

Drought stress triggers several plant responses at the gene expression levels, and likely result in the accumulation of secondary metabolites or osmolytes that help the plant stand the stress (Ramchandra Reddy et al. 2004, Ergen et al. 2009). In the present study, a large number of enzymes were found repressed due to watering, thus likely participate in the molecular mechanisms utilized to adapt to the long-lasting drought stress. Enrichment of these enzymes is not required when water becomes available, while re-enriched when land became dry again. KEGG analysis indicated that several gene families are involved as a safeguard against drought stress. Of these gene families, abscisic acid (ABA) receptor PYL seems to be required under drought stress for ABA-mediated responses such as stomatal closure (Hao et al. 2011). Suppression of two types of this receptor, namely PYL4 and PYL9 in senna (C. angustifolia) (Table 3, Figure 1), indicates that they participate in morphological changes as a mechanism of avoidance tolerance. PYL4 was reported to improved ABA-dependent inhibition of PP2CA and can modulate inhibition even in the absence of ABA (Pizzio et al. 2013). PP2CA plays a critical role to regulate both seed and vegetative responses to ABA and regulates stomatal aperture through interaction with the anion channel SLOW ANION CHANNEL1 (SLAC1) and the kinase OPEN STOMATA1 (Kuhn et al. 2006, Yoshida et al. 2006, Lee et al. 2009). In addition, ABA receptor PYL9 was proven to promote drought resistance and leaf senescence in Arabidopsis and rice (Zhao et al. 2016).

Figure 1: Enrichment pattern of the abscisic acid (ABA) receptors PYL4 and PYL9 at midday before (N) and after (NR) watering.   Click here to view figure

Auxin response factor (ARF) 5 and 15 are among the mechanisms of senna (C. angustifolia) to tolerate drought stress (Table 3, Figure 2). ARF are potential mediators of biotic and abiotic stress responses in plant (Bouzroud et al. 2018). ARF5 gene was proven to increase the contents of carotenoids and enhance the tolerance to both salt and drought when expressed in Arabidopsis (Kang et al. 2018), while ARF15 regulates cell division activity during early tomato fruit development, thus, provides important influence on plant growth under drought stress (DeJong et al. 2015). ARF5 gene also act as an activator of auxin-responsive gene expression in Arabidopsis (Remington et al. 2004). This enzyme allows the activation of ARF, derepresses downstream auxin responsive pathways, thus mediates plant growth and development (Gray et al. 2001). Also, two ARF (or Aux/IAA) proteins, namely IAA7 and IAA14, exist in stressed plant only (Table 3, Figure 3).

Table 3: Selected pathways of C. angustifolia with enzymes encoded by drought-related genes highly (≥5 FC) suppressed due to the watering. N = before watering, NR = after watering.

Figure 2: Enrichment pattern of auxin response factor (ARF) 5 and 15 at midday before (N) and after (NR) watering.   Click here to view figure

Figure 3: Enrichment pattern of AUX/IAA factors IAA14 and IAA7 at midday before (N) and after (NR) watering.   Click here to view figure

Aux/IAA proteins orchestrate several biological and physiological processes such as embryogenesis, leaf expansion and senescence, lateral root development and fruit development by regulating the expression of auxin response genes (Wilmoth et al. 2005, Sagar et al. 2013). IAA7 controls the morphological responses induced by light, e.g., promoting leaf development under adverse condition, while IAA14 acts in controlling lateral root formation by interacting with two ARF proteins (Luo et al. 2018). These two Aux/IAAs are produced due to the accumulated IAA generated is a result of tryptophan metabolism pathway and specifically oriented towards the induction of plant hormone signal transduction pathway to help the plant maintains normal performance inder drought stress. As the highly conserved domain II of the other Aux/IAA proteins is a target for degradation process promoted by auxin, this action allows the participation of the auxin-induced Protein TRANSPORT INHIBITOR RESPONSE 1 (TIR1). Consequently, TIR1 activates its target factors, e.g., ARFs, thus, allowing the auxin-responsive downstream genes, AUX/LAX, LBD and SAUR, to function and promote plant growth under adverse conditions. This Protein TRANSPORT INHIBITOR RESPONSE 1 (TIR1) also shown to be a major player under drought stress in senna as it is enriched only under the stress (Table 3, Figure 4). BRASSINOSTEROID INSENSITIVE 1 (BRI1)-associated receptor kinase 1 (or BAK1) belongs to a large group of plant transmembrane proteins known as the leucine-rich repeat receptor kinases (Belkhadir and Jaillais 2015). BAK1 has important role in brassinosteroid signaling (Li et al. 2002). Brassinosteroid is a plant hormone with important roles in cell proliferation and growth (Belkhadir and Jaillais 2015). BAK1 particularly acts in repressing the development of unneeded stomata in plant leaves, thus maintain water turgor under drought stress (Smakowska-Luzan et al. 2018). BAK1 also shown to be a major player under drought stress in senna as it is enriched only under the stress (Table 3, Figure 5).

Figure 4: Enrichment pattern of Protein Transport Inhibitor Response 1 (TIR1) at midday before (N) and after (NR) watering. Details of enrichment pattern of this protein is shown in Table 3.   Click here to view figure

Figure 5: Enrichment pattern of BRI1-associated receptor kinase 1 (BAK1) at midday before (N) and after (NR) watering. Details of enrichment pattern of this enzyme is shown in Table 3.   Click here to view figure

In senna, it is evident that PYL and BAK1 are induced by ABA and brassinosteroid towards the occurrence of stomatal closure and in cell elongation and division. These roles represent two bottle-necks in the plant hormone signal transduction pathway. Over and above, indole-3-pyruvate (or flavin) monooxygenase, encoded by YUC2 gene, also participates in tryptophan metabolism pathway (Watanabe and Lam 2006) towards the biosynthesis of IAA that, as mentioned above, is required for triggering the plant hormone signal transduction pathway. Accordingly, this enzyme likely has a role in conferring drought stress tolerance in senna (C. angustifolia) (Table 3, Figure 6). The two pathways likely crosstalk in senna (C. angustifolia) towards the maintenance of normal growth under adverse condition (Figures 7 and 8).

Figure 6: Enrichment pattern of indole-3-pyruvate (or flavin) monooxygenase at midday before (N) and after (NR) watering. Details of enrichment pattern of this enzyme is shown in Table 3.   Click here to view figure

Figure 7: Tryptophan metabolism pathway indicating two important enzymes Aldehyde dehydrogenase family 3 member F1 (EC: 1.2.1.3) and indole-3-pyruvate monooxygenase (EC: 11.41.31.68) that were enriched under drought stress, while suppressed due to watering. Details of enrichment pattern of these two enzymes are shown in Table 3.   Click here to view figure

Figure 8: Plant hormone signal transduction pathway indicating the auxin response factor  (ARF) and the Aux/IAA that were enriched under drought stress, while suppressed due to watering. Details of enrichment pattern of these two enzymes are shown in Table 3.   Click here to view figure

Phosphoinositide phosphatase SAC1 and SAC6 can also be among the molecular mechanisms to cope with the stress in senna (C. angustifolia) (Table 3, Figure 9). The domains of SAC protein possess phosphoinositide phosphatase activities. SAC1 was previously shown to affect diverse cellular functions such as normal cell morphogenesis, Golgi function, and maintenance of vacuole morphology (Zhong et al. 2005), while SAC6 gene was predominantly expressed in flowers and proven to be highly induced by salinity in Arabidopsis (Zhong and Ye 2003).

Figure 9: Enrichment pattern of SAC1 and SAC6 at midday before (N) and after (NR) watering. Details of enrichment pattern of these proteins are shown in Table 3.   Click here to view figure

A number of five pre-mRNA splicing factors as well as three serine/arginine-rich (SR) regulator factors were shown to participate in drought stress tolerance in senna (C. angustifolia) (Table 3, Figure 10). The pre-mRNA factors included splicing factors (SF) 8, 8A, 19, 40A and ISY1, while SRs included factors SCL33, RS31 and RS34. No particular function is assigned to any of these splicing factors and regulators except that each one might act on a certain protein or protein type. This conclusion warrant further experimentation and analysis. Pre-mRNAs usually contain introns that requires splicing or alternative splicing (AS) to produce structurally and functionally different proteins from the same gene (Palusa et al. 2007). Pre-mRNAs-related genes encode serine/arginine-rich (SR), which is a conserved protein family of splicing regulators in plant. Splicing of SR genes has proven to be developmental- and tissue-specific. Prior research indicated that abiotic stresses regulate the splicing of the pre-mRNAs of SR genes to produce different protein isoforms, thus different functions. We speculate that the altered splicing in senna seems like the action of the immune systems as to produce a specific antibodies for a specific antigen. Here, we think that alternative splicing of SR under stress produce the proper isoform of the proteins that can hold their structures and avoid denaturation or unfolding as a response to stimuli.

Figure 10: Enrichment pattern of the five Pre-mRNA splicing factors SF19, SF A40, SF 8, SF 8A and SF ISY1 as well as the three serine/arginine-rich SR34, SC35 and SR31 regulator factors at midday before (N) and after (NR) watering. Details of enrichment pattern of these proteins are shown in Table 3.   Click here to view figure

Many other regulated metabolites and proteins in senna (C. angustifolia) including protective proteins, such as heat shock protein 95s were previously reported to play functional roles in drought tolerance in plants (Hu and Xiong 2014, Umezawa et al. 2006). Senna might also developed efficient signal transduction cascades to cope with drought stress as it proved to regulate ATPase, which is a major signaling factor involved in drought stress signaling (Akpinar et al. 2012, 2013). Protein kinases participate in developmental and environmental signal transduction in plants (Rodriguez et al. 2010, Liu et al. 2016) and play a key role in activating transcription factors and drought-responsive proteins under drought tolerance. Among them, mitogen-activated protein kinase (MAPK) and cytochrome c oxidase that were regulated in senna (C. angustifolia) (Table 3).

Other enzymes include phospholipase C2 and allene oxide cyclase as well as isochorismate pathway were regulated in senna (C. angustifolia). The two enzymes play a role in JA pathway, while isochorismate pathway results in the production of salicylic acid (SA) (Kawano et al. 2004, Mustafa et al. 2009). The latter pathways have important applications in plant production.

In conclusion, we speculate that we have expanded our understanding of the molecular mechanism underlying drought stress tolerance in senna (C. angustifolia). This information will be valuable resource for accelerated genomics-assisted genetic breeding programs targeting the improvement of drought tolerance in economically important crop plants.

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