Emerging tools, concepts and ideas to track the modulator genes underlying plant drought adaptive traits: An overview

Abstract

Crop vulnerability to multiple abiotic stresses is increasing at an alarming rate in the current global climate change scenario, especially drought. Crop improvement for adaptive adjustments to accomplish stress tolerance requires a comprehensive understanding of the key contributory processes. This requires the identification and careful analysis of the critical morpho-physiological plant attributes and their genetic control. In this review we try to discuss the crucial traits underlying drought tolerance and the various modes followed to understand their molecular level regulation. Plant stress biology is progressing into new dimensions and a conscious attempt has been made to traverse through the various approaches and checkpoints that would be relevant to tackle drought stress limitations for sustainable crop production.

Introduction

Over 10,000 years ago humans initiated crop improvement through selective breeding to meet the increasing demand for food and fodder. Natural calamities and anthropogenic activities are posing serious challenges for sustained increase in global food production. Under present crop growing conditions, detrimental environmental factors, generally termed as abiotic stresses (drought, salinity, temperature extremes, nutrient deficiencies and toxicities) pose threat to normal growth and development of crop plants. These abiotic stresses can reduce average yield by more than 50%.^1-2 In tropical countries, although we come across multiple stresses simultaneously in crop lands, drought is considered as the major one.³ One-third of the world's population dwells in water-stressed regions and global climate change will definitely increase the occurrence and severity of drought episodes. Under the conditions of climate change and diminishing water resources crop biologists have the challenging task to evolve crop types that can grow and produce sufficient biomass and yield. Although attempts have been made by agronomists to mitigate drought stress threats, plant biologists/physiologists are interested in adopting a holistic approach of maintaining major biological processes under drought or desiccation stress. The ultimate aim should be to develop a genotype with commendable survival and production capacity under drought, rather than just survival characters, characteristic of xeric habitat adaptability. For identifying such characters, we need to overlook the various acclimatory processes that the plant adopts in order to equilibriate with the environment to reach the meta stable state called homeostasis, under unfavourable conditions.⁴ This homeostatic state is attained through various time dependent adaptive responses which make certain hardy species to withstand harsh detrimental conditions. This helps them to integrate various physiological processes in a coordinated manner to contribute to the greatest plant attribute called environmental plasticity.

The physiological integration of various tolerance/ acclimation mechanisms is a resultant of altered gene expression and/or existing protein functions. These responses pave way for either avoidance which prevent the very exposure to stress itself or tolerance strategies which permits the plant to thrive through the stress. Multiple factors govern stress tolerance including stress characteristics (duration, rate, severity and combinatorial effects) as well as plant characters (variations at genotype, developmental stage and tissue levels) which ultimately deliver a tolerant or susceptible phenotype. It is highly challenging to tackle the multi-threat face of drought, how and where to begin to strategise to achieve the goal of attaining tangible levels of drought tolerance.

How to Unravel the Complexity of Drought Stress Response: Trait Based Approach?

Nature's way of response to stress in most biological systems is the 'fight' or 'flight' response for every 'fright'. As a result of this phenomenon, plants being sessile, try to maintain essential metabolic activity for their sustenance and the degree or extent of response depends on different inherent tolerance associated mechanisms. The adaptive strategies revolve around certain key attributory physiological traits.⁵ Analysis of adaptive mechanisms of plants will contribute to the knowledge, the requirement for targeted crop improvement toward drought resistance. The complex responses to drought, from perception to ultimate physiological changes, need to be considered at a global systems biology level to examine the multiple interactive components.^6-7 Since drought tolerance is a complex trait, for targeted crop improvement, an understanding of the traits linked to plant water relations and cell tolerance to drought assumes significance and integrative traits are being used in high throughput phenotyping.^5,8 A few critical traits related to water relations such as water mining and water conservation determining water use efficiency (WUE) and cellular tolerance (CT) to desiccation are considered to be decisive for drought adaptation. The efficiency of the system to combat stress effects relies on how best the water relations are maintained either by mining more available soil water or by conserving water or both, along with various CT mechanisms. Efforts have been made to dissect and manipulate some of these characters with varied degrees of success.⁹

Water mining forms an important strategy wherein in the root system architecture attributes to tolerate drought to a considerable extent. Crops such as wheat with a deeper root system can have higher yields in rain fed systems due to efficient water mining ability.¹⁰ Improvement in root branching and density can lead to drought tolerance as noticed in rice using transgenic approach.¹¹ Many other root attributes have been reviewed extensively in the recent past.^12-13 There are also recent evidences emphasizing the importance of combining water acquisition and CT traits for maintaining higher spikelet fertility in rice under drought stress thereby enhancing field level tolerance to water limitation.¹⁴ In addition to water extraction from drying soil, water conservation strategies to retain tissue water are crucial for drought adaptation. Water conservation depends upon stomatal and non-stomatal transpiration, among which the later seems to be the crucial component since cuticular transpiration can happen continuously during day and night under dry conditions where vapour pressure deficit (VPD) will be high. In higher plants, cuticular wax forms a hydrophobic layer covering aerial organs, which is deposited either outside of the cuticle (epicuticular wax), or within the cuticular matrix (intracuticular wax).¹⁵ Epidermal wax layer provides a protective barrier between the plant and its environment, which functions as a barrier to water loss and prevents dehydration of underlying cells.¹⁵ Therefore, an important morpho-physiological feature (dehydration avoidance mechanism), the deposition of epicuticular waxes, can enable the plant to maintain hydration under the conditions of low VPD.⁸ However, reduction in transpiration can affect the evaporative cooling which makes it essential for a tradeoff between water conservation and stomatal regulation to bestow a cooler canopy. The genomic regions for canopy temperature in wheat have been identified recently and their genetic associations with stomatal conductance and grain yield have also been evaluated.¹⁶ A considerable number of such component traits are responsible for the quantitative regulation of utilization of water, the most critical resource under moisture deficit stress conditions, which could vary across diverse plant species and types.^17-18

Tissue water status determines the overall metabolic activity of the cells constituting it, which ultimately underlies the efficacy of various CT mechanisms. Cellular tolerance mechanisms equip each cell to build up a force against drought. Stress affects cellular energy status and induces energy saving responses resulting in low energy syndrome (LES). In general, LES includes transcriptional and translational reprogramming which is essential for stress acclimation.¹⁹ Cellular tolerance can be achieved by addressing key processes like transcriptional regulation, protein turnover, membrane stability attributory traits, active oxygen species scavenging (AOS) and so on.^7,9 Intrinsic cellular tolerance mechanisms can significantly contribute for growth and yield under abiotic stresses. Multiple cellular pathways and genes regulate cellular tolerance thereby offering a multi- level protection umbrella by employing various mechanisms like AOS scavenging, protein protection and turnover and osmoregulation depending on the species and genotype.²⁰ Rice plants overexpressing Arabidopsis homeodomain-leucine zipper transcription factor Enhanced Drought Tolerance/HOMEODOMAIN GLABROUS11 (EDT1/HDG11) had higher levels of abscisic acid, proline, soluble sugars and AOS-scavenging enzyme activities during stress which probably conferred drought tolerance and higher grain yield due to maintenance of pollen fertility.²⁰ These findings suggest that manipulation of cellular tolerance can immensely benefit plant growth and productivity under stress. Similarly, numerous traits including primary, secondary, and integrative drought-resistance traits exist, whose significance needs to be identified through precise phenotyping and be validated for intervention towards crop improvement for attainment of drought tolerance.^3,5,8 The molecular scrutiny of some of these traits is highly recommended. Hence the deployment of trait based gene prospecting assumes significance because of the fact that the current era is driven by 'gene revolution' for crop improvement after a yesteryear era of green revolution.

Prospecting the Trait Regulatory Genes

A wide array of gene discovery tools have helped to advance our understanding of stress signal perception and transduction and associated molecular regulatory networks.^21-23 These tools have helped in the successful revelation of several stress-inducible genes and various transcription factors that regulate the drought-stress-inducible systems. Initially, gene transcript analysis from drought tolerant crop species was adopted as a good approach for gene discovery, which has been primarily achieved by general gene expression analysis that is clearly reflected even in recent studies.^24-26 Later on, identification of the differentially expressed genes were successfully done by different approaches like subtractive hybridization, suppressive subtractive hybridization,²⁷ differential display,^28-29 cDNA-AFLP,³⁰ microarray technology and other means.^22,31 The microarray analysis of Arabidopsis thaliana genome has provided a powerful and widely used method to research the effects of various gene expressions which is also reflected in diverse plant species.³² Similarly, Targeting-Induced Local Lesions IN Genomes (TILLING), a reverse-genetic strategy for the discovery and mapping of induced mutations can also be attempted.³³ Generation and characterization of stress specific ESTs was attempted by many when genome information was limited.³⁴

Large-scale genome sequencing projects helped in the identification of important genes in certain plant types like Arabidopsis,³⁵ rice,³⁶ maize,³⁷ poplar³⁸ and other species, in the initial years. When the genome sequence is not available and genome size is significantly large, alternative approaches have been followed. Under such conditions, comparative genomics emerged as a viable attempt, which enables assessment of data from one species to investigate other incongruent species, which has already been achieved in case of annotating wheat sequences using information from E. coli, human, A. thaliana and rice.³⁹ However, in the recent past, the unravelling of genome information of a few species like the chickpea,⁴⁰ mulberry,⁴¹ soybean,^42-43 Medicago,⁴⁴ pigeon pea^45-46 and peanut (www.peanutbioscience.com)⁴⁷ has provided tremendous potential for targeted way of addressing various crop improvement issues. Over the years, technology advancement has led to a great leap in gene discovery from the mid 20th century to the late 2000s and till date (Fig. 1).

Figure 1.

Schematic depiction of the evolution of stress responsive gene discovery technologies over the years

The molecular breeding research ventures, in parallel, has helped in the identification of key quantitative trait loci (QTLs) defining the genomic regions and the underlying genes that regulate the various morpho-physiological traits that confer drought tolerance to superior accessions across different plant species.^48-54

Recently, a number of “omics” studies have reinforced the fact that the data resulting from these studies may promote our understanding of signaling pathways and will provide a new layer for analysis in systems biology.⁵⁵ Transcriptome based gene prospecting approach is gaining momentum these days.^56-57 Proteome and metabolome analyses in different plant species contributed to a better understanding of molecular mechanisms that are involved under drought.^58-60 There are several omics data repositories which are publically available as listed in Table 1, which can hasten our access to data generated for multi- level analysis for a comprehensive understanding of the experimental scenario. Integration of multiple data sets from different omics technologies have to be materialized by the use of various in silico and bioinformatics tools owing to the pressure of the rapid pace at which crop improvement programmes are advancing.^61-62 Whole plant tolerance attributes root deep within at individual cell/ tissue level which makes it highly significant to understand the cellular mechanisms of desiccation tolerance in diverse plants. This may eventually enable future molecular improvement for realizable levels of drought tolerance in crop plants.

Table 1.

List of publically available Omics Data Repositories

Repository	Web portal
DNA Data Bank of Japan (DDBJ)	http://www.ddbj.nig.ac.jp/
European Molecular Biology Laboratory (EMBL)	http://www.embl.org/
GenBank	http://www.ncbi.nlm.nih.gov/genbank
PlantGDB	http://www.plantgdb.org/
Gramene	http://www.gramene.org/
Rice Genome Annotation Project	http://rice.plantbiology.msu.edu/
Array Express	http://www.ebi.ac.uk/arrayexpress/
Gene Expression Omnibus (GEO)	http://www.ncbi.nlm.nih.gov/geo/
RiceXPro	http://ricexpro.dna.affrc.go.jp/
Maize Genome Database	http://www.maizegdb.org/
Botany Array Resource	http://www.bar.utoronto.ca/
PLANEX	http://www.planex.plantbioinformatics.org/
PLEXdb	http://www.plexdb.org/
EGENES	http://www.genome.jp/kegg-bin/create_kegg_menu?category=plants_egenes
Soybean database	http://www.proteome.dc.affrc.go.jp/Soybean/
Stress- Genomics	http://www.stress-genomics.org/
Plant Transcription Factor Database	http://planttfdb.cbi.pku.edu.cn/
Stress Responsive Transcription Factor Database V2.0	http://caps.ncbs.res.in/stifdb2/
Plant Promoter Database ver 3.0	http://ppdb.agr.gifu-u.ac.jp/ppdb/cgi-bin/index.cgi
Plant Proteome Database (PPBD)	http://ppdb.tc.cornell.edu/
Proteomics Identifications Database (PRIDE)	http://www.ebi.ac.uk/pride/archive/
Global Proteome Machine (GPMDB)	http://www.gpmdb.thegpm.org/
ProteomicsDB	http://www.proteomicsdb.org/
PeptideAtlas	http://www.peptideatlas.org/
PlaPID	http://www.plapid.net/
Plant Reactome	http://plants.reactome.org/
PlantMetabolomics.org	http://www.plantmetabolomics.org/
Plant Metabolome Database	http://www.sastra.edu/scbt/pmdb/
Plant Metabolic Network (PMN)	http://www.plantcyc.org/
MetNet online database	http://www.metnetonline.org/
MetNet	http://metnet.vrac.iastate.edu/
SIGnal	http://signal.salk.edu/
Plant Omics Data Center	http://www.bioinf.mind.meiji.ac.jp/podc/
Next- Gen Sequence Databases	http://mpss.udel.edu/
Phenome Networks	http://phnserver.phenome-networks.com/

Target Genes to Manipulate Cellular Tolerance-Stress Responsive Upstream Regulatory and Downstream Functional Genes

Stress responsive genes have specific elucidated roles that classify them under 2 major categories namely upstream regulatory genes and downstream regulatory/ functional genes.^22,63 However, the identity of the genes by proper annotation makes it meaningful in the context of its use for crop improvement. The rapid advancement in DNA sequencing technologies has accelerated plant biology research by unravelling the genomes of many important plant species keeping multiple ‘omics’ options open. This avenue is best realized when one gets to know the exact roles of the genes identified. The Gene Ontology (GO) concept has helped in the annotation of homologous gene and protein sequences in multiple organisms using a common platform which eases the query and retrieval of genes and proteins based on their shared biology.⁶⁴ A number of genes have been identified to be involved in CT, and their functions were confirmed by transgenic approaches.^2,7,11,65-67 It has also been reported recently that ectopic expression of stress specific transcription factors in combination aids in combating multiple abiotic stresses.^68-70 Large numbers of review articles have compiled the relevance of multiple upstream regulatory as well as downstream functional genes underlying a wide array of drought tolerance traits in imparting abiotic stress tolerance (Table 2). There is a well defined and tightly regulated signaling network starting from stress signal perception up to downstream functional gene activation, and the genes which regulate their activity, whichever be the tolerance mechanism.⁷¹ There have been several reports on the roles of different members in the signaling cadre which stand critical in imparting plant abiotic stress tolerance which is consolidated in Table 3.

Table 2.

Recent reviews on abiotic stress tolerance trait regulation in plants

Article content	Author and year of publication	Reference
Successful genetic engineering of drought-tolerant crops	Yang et al. 2010	81
Progress studies of drought-responsive genes in rice	Hadiarto and Tran, 2011	82
Targeting regulatory networks for abiotic stress tolerance	Reguera et al. 2012	67
Stress-induced metabolic rearrangements and regulatory networks	Krazensky and Jonak, 2012	71
Interaction of plant biotic and abiotic stresses	Atkinson and Urwin, 2012	83
Genetic engineering: evaluation of achievements, limitations, and possibilities	Lawlor, 2013	7
Recent advances in drought stress tolerance research in wheat	Rana et al. 2013	84
Genetic engineering and breeding of drought-resistant crops	Hu and Xiong, 2014	9
Metabolic adjustment and regulation of gene expression	Bhargava and Sawant, 2014	85
Transcriptional regulatory network in the drought response and its crosstalk	Nakashima et al. 2014	63
Safety aspects of genetically modified crops with abiotic stress tolerance	Liang et al. 2014	86
Drought-stress regulatory networks and strategies for drought-tolerant transgenic rice development	Todaka et al. 2015	87

Table 3.

Signaling partners associated with diverse abiotic stress responses in plants.

Signalling Cadre	Gene	Reference
Signal perception	ARCK1 (RLCK)	88
	GbRLK (RLK)	89
	GUDK (RLCK)	90
Signal transduction by phosphorelay	MAPKKK	91
	PtMKK4	92
	SpMPK3	93
	AtMPK12	94
	CBL-CIPK	95
		96
	CDPK1	97
Upstream regulatory genes
Stress specific transcription factors	NAC	98,105
	AP2/ERF	99,105
	WRKY	100,105
	HSFs	101,105
	bZIP	102,105
	Zinc fingers (ZF)	103,105
		104,105
Basal regulatory genes	BTF3	106
	NF-Ys	65,107,108
		107
		108
	TAFs-AtTAF10	109
Downstream regulatory/ functional genes
Water mining	GmNAC	110
	V-H+Ppase- ThVPV-H+Ppase- TaVP	111,112
Water conservation	EsWAX1	113
	DEWAX	114
Water relations	OsLEA3-2	115
	HVA1	116
Cellular tolerance
Osmoregulation
Trehalose	OsTPS1	117
	AtTPPD	118
Proline	P5CS	119
	PvP5CS	120
Mannitol	mtlD	121
Glycine betaine	codA	66
	codA	122
ROS scavenging	Ec-apx1	123
mRNA and Protein turnover	OsRDCP1	124
	OSRIP18	125
	CspA/B	126
	AtCSP3	127
	OsSUV3	128
	p68 helicase	129

Insights into Drought Responsive Gene Regulation are Critical

Drought being an oligogenic trait, has to be addressed by encompassing multiple genes wherein understanding the regulatory attributes of the activity of the gene in question would be the most critical aspect. In this direction, regulon biology is catching up in the race as a new area in the field of plant molecular stress biology.^72-73 Regulons are thought to be key regulators which have an influence on the expression of multiple effector genes. Regulons are a group of operons/genes spread around the chromosome but controlled by a common factor or stimulus and multiple regulons form a modulon. There are many transcriptional switches that regulate various plant processes at different developmental stages;⁷⁴ however, the similar information under diverse environmental conditions is limited.⁷⁵ There have been recent reports on this emerging concept. A regulon conserved in monocot and dicot plants defines a functional module in antifungal plant immunity.⁷⁶ DUO1 regulon encompasses genes with a range of cellular functions, including transcription, protein fate, signaling, and transport in Arabidopsis. Hence, Arabidopsis DUO1 regulon has a major role in shaping the germline transcriptome and functions to commit progenitor germ cells to sperm cell differentiation.⁷⁷ In this direction, a concerted effort in identification of DNA regulatory motifs assumes significance,⁷⁸ since this will help in recognizing the crucial regulons contributing to stress responses. Hence, characterization of plant responses to multiple stress conditions and discovery of the common regulons activated under a variety of stress conditions is very vital. In addition to the knowledge gained on conserved regulatory motifs, it is very essential to understand the importance of certain domains of unknown function found in many novel proteins enriched under stress with respect to their binding specificity.^79-80 This in turn will help in the unravelling of various interactive partners that aid specific proteins in their mission of stress protection under each stress.

Conclusion

Diverse genes linked to various cellular tolerance mechanisms activated under drought stress act in a concerted manner to bestow varying degrees of stress tolerance. It will be highly rewarding if we examine different pathway linked genes active in the stress scenario under scrutiny. Targeted genetic manipulation to enhance cellular tolerance under stress will be more economically viable if we combine multiple trait regulatory genes by using modern biotechnological tools. This approach will serve in managing drought tolerance which is a complex multi-trait faceted attribute, which is the key to higher marginal productivity under stress.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by University Grants Commission-Council of Scientific and Industrial Research, Government of India in the form of Junior Research Fellowship to PMS (File No. F.17–3/2002(SA-I) and partial funding from the Department of Biotechnology and Indian Council of Agricultural Research, Government of India to KNN.