Current status of the production of high temperature tolerant transgenic crops for cultivation in warmer climates.

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PLAPHY4101_proof ■ 23 November 2014 ■ 1/9

Plant Physiology and Biochemistry xxx (2014) 1e9

Contents lists available at ScienceDirect

Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy

Review

Current status of the production of high temperature tolerant transgenic crops for cultivation in warmer climates Q5

Dhruv Lavania a, Anuradha Dhingra a, Manzer H. Siddiqui b, Mohamed H. Al-Whaibi b, Anil Grover a, * a b

Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi 110021, India Department of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 October 2014 Accepted 20 November 2014 Available online xxx

Climate change is resulting in heightened incidences of plant heat stress episodes. Production of transgenic crops with enhanced heat stress tolerance is a highly desired agronomic trait for the sustainability of food production in 21st century. We review the current status of our understanding of the high temperature stress response of plants. We specifically deliberate on the progress made in altering levels of heat shock proteins (Hsp100, Hsp70/Hsp40 and sHsps), heat shock factors and specific metabolic proteins in improving plant tolerance to heat stress by transgenic approach. © 2014 Published by Elsevier Masson SAS.

Keywords: Heat shock factors Heat shock proteins Heat stress Thermotolerance Transgenics

1. Introduction

Q2

Largely, as a result of anthropogenic factors, Earth's ambient temperature has been slowly rising. Of late, this process has gained momentum. Owing to climate change and global warming, the increase in global mean surface temperature is projected to be in the range of 1.5e4 C by the end of the 21st century (IPCC, 2013). Climate change is causing frequent fluctuations in daily and seasonal temperatures with increased and prolonged incidents of heat waves. Field-plants are experiencing increased levels of heat stress both during day (high day temperature stress) and night (high night temperature stress). The yields of the major world food crops are already on the decline due to heat stress (Lobell and Gourdji, 2012; Teixeira et al., 2013). Temperate and sub-tropical agricultural zones are in particular more prone to yield losses due to extreme temperature regimes (Teixeira et al., 2013). Critical reproductive stages are threatened by high temperature stress because of changes in crop calendars (Teixeira et al., 2013). The sensitivity of the crops to heat stress varies: some species and cultivars being more sensitive to heat stress than others (Lobell and

* Corresponding author. E-mail addresses: [email protected] (D. Lavania), dhingra.anu5@gmail. com (A. Dhingra), [email protected] (M.H. Siddiqui), [email protected] (M.H. Al-Whaibi), [email protected] (A. Grover).

Gourdji, 2012). It is possible that the sensitive plants may somewhat adapt to heat stress naturally in due course. The pollen grains of heat tolerant cultivars in some crops constitutively express the specific thermoprotective genes at higher levels, as against the heat sensitive cultivars (Bita et al., 2011). However, the capacity of plant species to evolve naturally against temperature fluctuations is a slow process. Urgent measures are required to combat the effects of high temperature stress on global crop productivity to sustain food security in the foreseeable future. Genetic improvement of plants to tolerate heat stress could be a way to achieve this goal. The conventional plant breeding methods have worked effectively for breeding resistance against several biotic and abiotic stresses. However, this approach, by and large, has not been highly successful against abiotic stresses (including heat stress) because of the complex genetics of the involved biochemical/physiological mechanisms. Marker-assisted breeding has been successful against some abiotic stresses (i.e. salt stress, flooding stress). However, its application against heat stress has not been reported as yet. The transgenic plant production approach has emerged as a powerful tool for addressing numerous agronomic traits including breeding increased resistance against biotic and abiotic stresses. We present the salient features of the work carried out for producing heat tolerant transgenic plants. We also discuss the road ahead in this important endeavor. More specifically, we focus on the roles of heat shock proteins (Hsps)/heat shock factors

http://dx.doi.org/10.1016/j.plaphy.2014.11.019 0981-9428/© 2014 Published by Elsevier Masson SAS.

Please cite this article in press as: Lavania, D., et al., Current status of the production of high temperature tolerant transgenic crops for cultivation in warmer climates, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.11.019

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D. Lavania et al. / Plant Physiology and Biochemistry xxx (2014) 1e9

Table 1 Selected details on genome-wide studies undertaken to analyze the heat stress response in recent years. HS: heat stress. Plant species [platform employed]

Salient changes in gene expression

Significant variations in HS responsive (38 C; 3 h) transcriptional changes in 10 A. thaliana ecotypes from different geographical origins. 3644 transcripts significantly regulated by HS in the 10 ecotypes consisting of 244 transcription factors and 203 transposable elements. 31 Hsps showed ecotype specific profiles under HS. Ecotype specific expression patterns of 35 HS responsive transcription factors. Hordeum vulgare [Affymetrix Of the 2078 differentially 22K Barley1 GeneChip] expressed genes in H. vulgare caryopses, 956 and 1122 genes were up-regulated and downregulated, respectively at one or more HS time points (42 C; 0.5, 3 and 6 h). About 204 core HS response gene (3 Hsfs, 27 Hsps and 14 chaperonins and DnaJ) identified. Expression of genes for starch biosynthesis was repressed and for starch degradation was elevated under HS. 141 endosperm specific and 28 embryo specific genes were HS responsive. Transcriptome profiling of rice O. sativa cv. Pusa Basmati 1 [Agilent 60 mer rice 44 K oligo seedlings subject to sub-lethal HS (42 C; 10 min and 30 min). DNA array] Transcript levels of genes for Hsps, Hsfs, ROS metabolism, sugar and auxin metabolism, RNA metabolism significantly modulated. At HS 10 min, 557 genes exclusively up-regulated and 288 genes exclusively downregulated. At HS 30 min, 222 genes exclusively up-regulated and 295 genes exclusively downregulated. ROS homeostasis identified as pivotal process in HS response with 36 upregulated genes common to HS and oxidative stress. O. sativa cv. 996 [Agilent 4 44 K Young rice florets at anther rice oligo microarray] development stage 8 used for microarray profiling under time course of HS (40 C; 0 min, 20 min, 1 h, 2 h, 4 h and 8 h). 2449 genes identified as HS responsive comprising transcription factors (Hsfs, AP2/ ERFs, WRKYs, NACs, bZIPs, bHLHs, MYBs and C2H2), Hsps (Hsp100, Hsp70, Hsp90, sHSP, Hsp60, Hsp40, Hsp20, chaperonins and DnaJ proteins), transporters genes (secondary transporters, ion channels, ATPdependent transporters), ROS homeostasis genes (P450 family, glutathione peroxidase, peroxiredoxin, thioredoxin), genes involved in auxin signaling, ethylene metabolism and ABA metabolism. O. sativa cv. 996 [Agilent 4 44 K Tanscriptome profiling of rice rice oligo microarray] flag leaf under time course of HS A. thaliana [Arabidopsis NimbleGen ATH6 microarrays]

Reference Barah et al. (2013)

Mangelsen et al. (2010)

Mittal et al. (2012)

Zhang et al. (2012)

Zhang et al. (2013)

Table 1 (continued ) Plant species [platform employed]


(40 C; 0 min, 20 min, 30 min, 1 h, 2 h, 4 h and 8 h) showed more than 3-fold up-regulation of 4153 genes. These comprised genes for transcription factors (Hsf, AP2/ERF, bHLH, bZIP, MYB, WRKY, NAC and C2H2), Hsps, ROS metabolism, transport, glycolysis, ubiquitin proteasome system (E2 and E3 ubiquitin ligases), rate-limiting enzymes of shikimate, lignin and mevalonic acid metabolism. Downregulation of genes for carbohydrate metabolism, carotenoid, flavonoid and anthocyanin metabolism and light reaction genes. Profiling of transcriptional O. sativa cv. Pusa Basmati 1 [GreenGene Biotech 60 K rice events in early HS response (42 C; 10 min and 60 min) and whole genome microarray] recovery (26 C; 30 min after 42 C; 60 min). Post 10 min HS, greater number of up-regulated genes (1556), comprising signal transduction, transcriptional control and ROS metabolism genes, than down-regulated genes (600) and vice versa post 60 min HS (2064 upregulated and 2466 downregulated). 452 genes upregulated in all three treatments Gene expression profiles of S. lycopersicum; [90 K Custom meiotic anthers of three heat TomatoArray 1.0 chip sensitive genotypes (Money(Combimatrix microarray maker, Falcorosso and Pull) platform)] compared with two heat resistant genotypes (Heat Set1 and Saladette) under moderate HS (32 C/26 C, day/night; 0, 2, 6, 16 or 30 h). Genotypic differences in gene expression observed in terms of number and level of expression changes. Moderate expression changes (general trend of up-regulation) and constitutively high expression of stress protection genes in tolerant genotypes as against general trend of downregulation in sensitive genotypes. Major differentially expressed genes belonging to Hsps, signal transduction, transcription factors, ROS metabolism, cell transport, carbon metabolism and development. e [custom Eight week old plants grown S. tuberosum cv. Desire Agilent microarray platform under moderately elevated temperature (30 C/20 C; day/ 60 mer 8 60 K format] night; 0, 4, 8, 12, 16, 20 h). 2190 differentially expressed genes in leaf comprising overrepresentation of functional categories for heat response, photosynthesis, lipid metabolism, amino acid biosynthesis and hormone metabolism. In tubers, differentially expressed RNA metabolism genes were overrepresented whereas, categories

Reference

Sarkar et al. (2014)

Bita et al. (2011)

Hancock et al. (2014)


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2. Preparedness of plants to heat stress

Table 1 (continued ) Plant species [platform employed]

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for cell wall processes, lipid metabolism, secondary metabolism, hormone metabolism, biotic stress, DNA metabolism and development were under-represented. Sorghum bicolor Moench variety 4% (966 up-regulated and 224 down-regulated), 18% (2765 upR16 [custom Agilent microarray platform 60 mer regulated and 2406 downregulated) and 20% (3003 up4 44K format] regulated and 2776 downregulated) transcripts differentially expressed after drought stress, HS (50 C; 3 h) and combination of the two stresses (3 d post water withdrawal; 50 C). Many of the transcript changes were common but 7% were unique to stress combination. Only HS responsive, differentially expressed genes include, Hsps, universal stress proteins and antioxidants. Expression changes common to both stresses include genes for heat shock proteins, senescence-associated genes and glutathione transferases. Heat susceptible and tolerant Triticum aestivum [Affymetrix genotypes, Chinese Spring and GeneChip Wheat Genome TAM107, respectively subject to Array] HS (40 C; 1 h and 40 C; 24 h) with and without heat acclimation (34 C; 3 h). 6560 probesets (~10.7%) showed 2fold or more up-regulation and differential expression in the two genotypes (313 probe sets between the two genotypes). These majorly include genes for Hsps, Hsfs, transcription factors, ribosomal proteins, RNA metabolism, phytohormone biosynthesis/signaling, calcium and sugar signaling, primary and secondary metabolism and other stress related genes. Vitis vinifera [NimbleGen 090818 Short and abrupt HS Vitis 12 30 K microarray] administered at day and night (37 C; 2 h after sunrise and 2 h after sunset) to different developmental stages of grapevine berries. Transcriptome profiles varied considerably between day and night in stagespecific manner. During day stress, 1154 genes were upregulated and 1638 were downregulated and during night stress, 2455 genes were upregulated and 2142 genes were down-regulated in at least one of three developmental stages and time points. Higher gene expression changes were induced by high night temperature stress.

Reference

Johnson et al. (2014)

Qin et al. (2008)

Rienth et al. (2014)

(Hsfs) circuitry and specific metabolic activities in governing the plant heat response. To keep the ‘References’ list short, we are including only the selected papers; readers may cross-refer (Grover et al., 2013) for more detailed reference list.

Plants employ both short-term and long-term adaptations to survive under stressful conditions. These adaptations help the plants to escape the stress (heat avoidance) or to develop mechanism(s) such that the metabolic reactions/processes show a sustained activity even under stressful conditions (heat tolerance) (Bita and Gerats, 2013; Hasanuzzaman et al., 2013). Avoidance related specialized adaptations (like shorter life cycle, development of morphogenic structures to protect heat sensitive tissues etc.) are usually constitutively expressed and relatively complex in nature and thus require multiple genes to be formed. The simple and limited gene transfers carried out in transgenic plants may not work in evolving the avoidance mechanisms. The tolerance mechanisms to heat stress are of induced type and on relative basis, simpler in terms of involved genetics. The heat tolerance-related mechanisms are more amenable to genetic alterations and have therefore been the focus of transgenic experiments. In case of model plant species Arabidopsis thaliana, heat tolerance has been classified as basal thermotolerance, short-term acquired thermotolerance, long-term acquired thermotolerance and thermotolerance to moderately high temperatures (Yeh et al., 2012). However, the detailed events associated with these four specific thermotolerance response types are yet to be firmly established. There is clear need to define such thermotolerance responses in other plant species as well. Supra-optimal heat levels affect diverse cellular reactions and processes. The molecular and physiological changes affected by heat stress vary significantly for tissues, growth stages and stress levels. These changes further vary among species and within the species, from variety to variety. The photosynthetic machinery components (i.e. photochemical electron transport activities, ribulose bisphosphate carboxylase/oxygenase activity, ribulose bisphosphate activase activity) and selective phenological stages (i.e. pollen development, anther dehiscence, pollen germination, fertilization process and grain filling) are particularly vulnerable to heat stress (Endo et al., 2009; Sakata and Higashitani, 2008; Young et al., 2004). High sensitivity of pollen to heat stress and resultant decline in yield has been noted across plant species (Endo et al., 2009; Harsant et al., 2013; Jagadish et al., 2010). Overall, the cellular processes associated with transcription and translation activities, reactive oxygen species (ROS) metabolism, membrane permeability, cytoskeletal reorganization, synthesis of osmolytes and protective secondary metabolites are critical in determining response of plants to high temperature stress. Selected recent investigations on high-throughput transcriptomic profiling of plants in response to heat stress are discussed in Table 1. From the account provided in Table 1, it is amply clear that heat stress affects the biological systems in a drastic way through both general repression and specific activation of a large number of genes. The transcripts significantly up-regulated under heat stress primarily encompass those of Hsps, Hsfs and enzymes responsible for the synthesis of osmolytes, membrane characteristics and ROS metabolism (Mittler et al., 2012; Sarkar et al., 2014). The perception of the heat signal is attributed mainly to four putative ‘heat sensors’ namely, cyclic nucleotide gated calcium channels on the plasma membrane, an unfolded protein response (UPR) sensor in the endoplasmic reticulum, a UPR sensor in the cytosol and a histone H2A.Z variant in the nucleus (Wang et al., 2012a,b). These components reportedly sense heat and trigger the signal transduction cascades leading to the expression of Hsp genes and thermotolerance. However, understanding of these sensors in terms of mechanism and order of their activation, stress- and tissue-specificity, inter-relatedness and conservation is far from complete. Besides the stressful temperatures, the wide range of non-stressful ambient temperature


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Table 2 Selected details of the transgenic plants raised against heat stress in recent studies. HS: heat stress, WT: wild type. Trans-gene (trans-protein)

Trans-host plant species; promoter employed

Trans-gene involved in membrane protection GPAT (glycerol-3-phosphate Nicotiana tabacum cv. NC89; acyltransferase) CaMV35S

Trans-gene involved in osmotic homeostasis BADH (betaine aldehyde T. aestivum cv. Shi4185; maize dehydrogenase) ubiquitin (Ubi1) COD A (choline oxidase)

COD A (choline oxidase)

Brassica compestris L. spp. chinensis var. Aikangqing; CaMV35S S. lycopersicum cv. Moneymaker; CaMV35S

Trans-gene involved in ROS metabolism GRXS17 (monothiol S. lycopersicum. cv. Rubion; glutaredoxin) CaMV35S

NTRC (NADPH-dependent thioredoxin reductase type C)

A. thaliana ecotype Columbia; CaMV35S

CKX1 (cytokinin oxidase/ dehydrogenase) Trans-gene involved in signal CBPK3 (calmodulin-binding kinase)

N. tabacum cv. Samsun NN; CaMV35S/WRKY6 transduction A. thaliana ecotype Columbia; CaMV35S

PP5 (serine/threonine protein phosphatase)

A. thaliana Heynh ecotype Columbia; CaMV35S

SGTL1 (sterol glycosyltransferase)


PLC3/PLC9 (phosphinositidespecific phospholipase C)


RCF2 (protein phosphatase)


Stress treatment parameters and phenotyping of the transgenic lines

Reference

Increased saturation of thylakoid membrane lipids of transgenic plants. Under HS (3e4 Yan et al. month old; 25e48 C; 4 h), photosynthesis parameters of photosystem II (PSII) decreased (2008) at a slower rate in transgenic plants compared to WT and showed faster recovery post stress. Glycinebetaine (GB) over accumulation in transgenics resulted in higher protection and stability of thylakoid membrane lipids and decreased photoinhibition of PSII under HS (40 C; 3 h), drought stress (30% polyethylene glycol) and combination of the two stresses. GB accumulation in transgenics resulted in higher net photosynthetic rate (Pn) and Pn recovery rate under HS (45 C; 4 h; 1 h recovery) and cold stress (1 C; 48 h; 1 h recovery). Higher Pn under high salinity (100, 200 and 300 mmol/L NaCl) stress. GB accumulation in transgenics improved seed germination and increased expression of Hsps in seeds after HS treatment. HS given during imbibition (25, 40, 50 and 55 C; 90 min, 25 C; 3e12 d), during germination (40 C/30 C; 12 h/12 h or 34 C/34 C; 16 h/8 h for 12 d) and during seedling growth (4 d old; 34 C; 7 d). Increased heat tolerance and better recovery of transgenics than WT (4 week old; 38 C/ 28 C day/night; 3 d, 42 C/32 C day/night; 3 d, 25 C/22 C) with no adverse effects on fruit size and shape. Transgenics showed reduced chlorophyll photo-oxidation and oxidative damage of cell membranes. Increased catalase activity and reduced H2O2 accumulation in over-expression transgenic plants. Enhanced acquired thermotolerance in over-expression transgenics (3 d old; 37 C; 1 h, 22 C; 2 h, 45 C; 2.5 h, 22 C; 7 d). Knockout mutant defective in acquired thermotolerance. Impaired basal thermotolerance in over-expression transgenics and knockout mutant (3 d old; 45 C; 20 min and 30 min, 22 C; 7 d). Lesser ion leakage induced by HS in transgenics. Higher relative water content in transgenic plants. Higher expression of several antioxidant enzymes in over-expression transgenics. Increased basal thermotolerance of transgenic plants (10 d old; 45 C; 45 min, 22 C; 7 d). Defective basal thermotolerance of knockout mutant rescued by complementation with WT gene. Up-regulated expresssion of Hsp genes in transgenics after HS (37 C; 1 h) and increased synthesis of Hsps after HS (14 d old; 37 C; 2 h). Increased basal thermotolerance of transgenics (7 d old; 55 C; 20 min, 22 C; 9 d). Increased basal thermotolerance in soil grown transgenics (14 d old; 37 C; 5 d, 22 C; 5 d). Knockout mutant defective in acquired thermotolerance (3 d old; 37 C; 60 min, 22 C; 120 min, 45 C; 160 min, 22 C; 5 d). Enhanced basal thermotolerance of plate grown (14 d old; 45 C; 180 min, 22 C; 7 d) and soil grown transgenics (21 d old; 42 C; 60 min, 22 C; 7 d). Enhanced acquired thermotolerance of plate grown transgenics (7 d old; 38 C; 90 min, 22 C; 120 min, 42 C; 180 min, 22 C; 5 d). Lesser heat induced malodinaldehyde (MDA) content/lipid peroxidation and higher expression of Hsp70 and Hsp90 in transgenics compared to WT. Transgenics tolerant to cold and salinity stresses also. plc3 knockout mutants showed lesser heat tolerance (7 d old; 45 C; 65 min, 22 C; 7 d) with 40e50% lower survival rate and chlorophyll content than WT. Mutant phenotype restored in complemented lines under HS. Increased survival rate and chlorophyll content of over-expression transgenics under HS (7 d old; 45 C; 75 min, 22 C; 7 d). plc3/plc9 double mutant more heat sensitive than single mutants. Expression of Hsp genes under HS (10 d old; 37C; 1 h) lower in plc3 knockout mutant and higher in over-expression transgenics. Knockout mutant more thermosensitive than WT (3 week old soil grown; 37 C; 7 d) and flowers of mutant more sensitive than WT flowers (1 month old; 37 C; 4 d). Reduced expression of HS responsive genes in mutant plants after HS. Higher thermotolerance in over-expression transgenic plants (5 week old soil grown; 37 C; 2 weeks) and flowers (5 week old soil grown; 37 C; 4 d). Higher expression of HS responsive genes after HS in total seedlings and flowers (15 d old or 1 month old; 37 C; 1 h).

Trans-genes involved in sumoylation and ubiquitination OsSIZ1 (SUMO E3 ligase) Agrostis stolonifera cv. Penn A-4; Higher shoot biomass, chlorophyll content and Pn of transgenic plants. Under HS (35 C/ maize Ubi1 30 C light/dark; 6 d, 40 C/35 C light/dark; 6 d, 20 C; 2 weeks), trasngenics displayed less reduction in root and shoot biomass, higher relative water content, improved root growth and lesser cell membrane damage as compared to WT. Increased HS induced SUMO conjugation to substrate proteins in transgenics. OsHCI1 (RING finger E3 ligase) A. thaliana ecotype Columbia; Increased acquired thermotolerance in over-expression transgenics (7 d old; 38 C; CaMV35S 90 min, 24 C; 120 min, 45 C; 3 h, 24 C; 5 d) with 55e65% survival rate BnTR1 (RINGv E3 ligase) B. napus and O. sativa; CaMV35S Over-expression resulted in HS tolerance in transgenic B. napus with higher survival rate (2 week old; 35C; 6 and 9 d) than WT. Over-expression in O. sativa increased HS tolerance (40 d old; 48 C; 30 min, 26 C; 3, 4 and 45 d) with no adverse effect on growth and development of transgenics. Ta-Ub2 (monoubiquitin) N. tabacum; CaMV35S Enhancement of photosynthesis under HS (4 month old; 45 C; 3, 6 and 9 h) in transgenics with higher D1 protein levels, Pn and maximal photchemical efficiency of PSII (Fv/Fm). Increased redox enzyme activities, lower lipid peroxidation and ROS accumulation under

Wang et al. (2010) Wang et al. (2010) Li et al. (2011)

Wu et al. (2012a,b)

Chae et al. (2013)

Lubovask a et al. (2014) Liu et al. (2008)

Park et al. (2011)

Mishra et al. (2013)

Gao et al. (2014)

Guan et al. (2014)

Li et al. (2013)

Lim et al. (2013) Liu et al. (2014)

Tian et al. (2014)


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Table 2 (continued ) Trans-gene (trans-protein)

Trans-host plant species; promoter employed

Stress treatment parameters and phenotyping of the transgenic lines

Reference

HS. Increased amount of ubiquitin conjugated proteins under HS suggesting increased degradation of HS damaged proteins. Trans-gene involved in transcription WRKY25, WRKY 26 and WRKY33 A. thaliana ecotype Columbia; (WRKY transcription factors) CaMV35S

JUB1 (ROS-responsive NAC transcription factor) OsMYB55 (R2R3 MYB transcription factor)

A. thaliana ecotype Columbia; CaMV35S O. sativa cv. Kaybonnet; Maize Ubi1

ERF1 (ethylene responsive transcription factor)


CBF3 (C-repeat binding factor)

S. tuberosum cv. luyin NO.1; CaMV35S/rd29A

Trans-gene involved in other processes LeUCP (mitochondrial S. lycopersicum cv. Ailsa Craig; uncoupling protein) CaMV35S

PpEXP1 (expansin)

N. tabacum; CaMV35S

TaLTP3 (lipid transfer protein)


Increased germination rate of over-expression transgenics after HS (45 C; 4 h). Enhanced thermotolerance of over-expression transgenics (25 d old; 48 C; 6 h, 22 C; 9 d). Single, double and triple knockout mutants exhibited various degrees of thermosenstivity of seed germination and seedling survival. Higher expression of HS responsive genes in overexpression transgenics post stress (21 d old; 42 C; 30 min and 60 min). Over-expression resulted in increased thermolotolerance (7 d old; 45 C; 45 min, 23 C; 8 d). Reduced thermotolerance of knockout mutant (2 week old; 45 C; 5 h, 22 C; 5 d). Longer coleoptiles of over-expression transgenics grown under HS (39 C; 5 d). Greater dry biomass, root dry biomass, plant height, leaf sheath length and grain yield of transgenics under HS (35 C/26 C day/night; 4 weeks, 29 C/23 C day/night; until harvest). Transcriptional activation of several amino acid metabolism genes during HS (4 week old; 45 C; 1, 6 and 24 h) resulting into increased total amino acid content in transgenic plants. Over-expression transgenic plants displayed increased basal thermotolerance (7 d old; 45 C; 1 h, 22 C; 7 d) and up-regulation of 32 heat responsive genes. Transgenics displayed marked tolerance to drought and salt stresses also. Higher Pn, Fv/Fm in over-expression transgenics under HS treatments (6 week old; 25, 30, 35, 40 or 45 C; 2 h and 40 C; 2 h or 4 h, 25 C; 24 h). Reduced ROS production, induction of several stress responsive and antioxidant genes after HS (40 C; 4 h, 25 C; 24 h). Transgenics exhibited increased growth rate under HS (42 C/38 C; 21 d) along with increased chlorophyll content, Fv/Fm, photochemical quenching coefficient and electron transport rate, ascorbic acid content, proline content, lesser ROS production under HS (42 C/38 C; 48 h) leading to elevated HS tolerance. Transgenics also tolerant to Botrytis cinerea infection. Increased protection of cell structure of transgenics under HS. Higher leaf membrane stability, relative water content, chlorophyll content, Pn, superoxide dismutase (SOD) activity and lower lipid peroxidation and H2O2 accumulation under HS (2 week old; 42 C; 6 d). Higher germination rate of transgenic seeds under HS (30, 40 and 50 C; 2 h). Basal thermotolerance of transgenics was enhanced higher survival rate (7 d old; 45 C; 2 h, 22 C; 4 d) and hypocotyl elongation under HS (3 d old dark grown; with or without pre-acclimation 38 C; 90 min, 45 C; 2 h, 22 C; 2.5 d). Lower HS induced electrolyte leakage (3 week old; 42 C; 1 h) and reduced ROS production under HS in roots (7 d old; 45 C; 1 h) and in rosette leaves (3 week old; 45 C; 1 h) of over-expression transgenics.

profoundly affects various aspects of plant growth and development, disease resistance and circadian clock in A. thaliana without the activation of heat shock response. Phytochrome interacting factor 4, a basic helix loop helix transcription factor, and H2A.Znucleosomes have been identified as critical components mediating changes in gene expression in response to fluctuations in ambient temperatures (Wigge, 2013). Of late, heat responsive small interfering RNAs (siRNA) such as micro RNAs and trans-acting siRNAs have been implicated in the regulation of thermotolerance pathways (Guan et al., 2013; Li et al., 2014; Lu et al., 2013; Wang et al., 2012a,b; Yu et al., 2012, 2013). In addition, genome wide epigenetic alterations such as RNA-directed DNA methylation, heterochromatin reorganization and changes in nucleosome composition affect heat stress response and stress memory in plants (Boden et al., 2013; Popova et al., 2013; Wang et al., 2013). Out of the plethora of gene expression changes associated with heat stress response as of above, four distinctive sets of proteins which have been targeted for production of heat tolerant transgenic plants include (1) Hsps as such or Hsfs which down-stream regulate Hsps, (2) enzymes involved in synthesis of osmolytes, (3) enzymes governing fluidity of membranes and (4) enzymes involved in maintenance of ROS levels. The detailed contribution of these proteins in heat response is discussed elsewhere (Grover et al., 2013) and an update of the same is presented in Table 2.

Li et al. (2011a,b)

Wu et al. (2012) El-kereamy et al. (2012)

Cheng et al. (2013) Dou et al. (2014)

Chen et al. (2013)

Xu et al. (2014)

Wang et al. (2014a,b)

3. Heat shock proteins and heat shock factors: plants' major arsenal against heat stress The homeostasis of cellular proteins (i.e. proteostasis) is disturbed when heat stress sets in. Protein structure is of paramount importance for the protein functioning. When protein structure gets affected by heat stress (in terms of denaturation, misfolding or aggregation), the protein functioning gets negatively altered. The mis-folded proteins prove toxic to cells, resulting in ‘diseased state’ in animal cells. In order to minimize damage to cellular proteins, cellular levels of chaperone proteins are generally up-regulated. As most Hsps act as chaperones, the expression of Hsps has been correlated with the acquisition of high temperature tolerance in numerous instances. Despite constituting a small portion of the thermal transcriptome of plants, the high level expression of Hsfs and Hsps stands out in the heat stress acclimation process (Mittler et al., 2012). Mutant cell lines in which Hsps are experimentally repressed often show high sensitivity to heat stress (Lin et al., 2014; Ruibal et al., 2013; Zhong et al., 2013). Accumulation of Hsps in early stages of pollen development was shown recently (Chaturvedi et al., 2013). Relative rates and levels of Hsps vary between and within different plant species. It has been rationalized that over-production of Hsps may lead to increased heat tolerance. Several experiments have been aimed at the overproduction of Hsps in plants (Grover et al., 2013). Plant Hsps are broadly classified as Hsp100, Hsp90, Hsp70/40, Hsp60/10 and sHsps classes. Hsp90 have important role in unstressed conditions and in attenuation period of heat shock response by regulating Hsf activity, either by direct interaction or


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by targeting Hsfs to proteasomal degradation. The Hsp90 gene family is comprised of seven genes in A. thaliana and twelve genes in soybean (Krishna and Gloor, 2001; Xu et al., 2013). In a recent study, over-expression of five Hsp90 genes of Glycine max in A. thaliana resulted in higher biomass production and pod setting and reduction in lipid peroxidation and loss of chlorophyll under heat stress (Xu et al., 2013). Bulk of the work on production of heat tolerant transgenic plants has focused on Hsp100, Hsp70/Hsp40 and sHsp classes. Hsp100 proteins belong to caseinolytic protease (Clp) family proteins, more specifically the ClpB family, representing class I ATPase group proteins. Clps carry out disaggregation of denatured proteins and are implicated in both basal and acquired thermotolerance (Grover et al., 2013). ClpB proteins of plants fall into 3 classes, namely, cytoplasmic/nuclear (ClpB-C), chloroplastic (ClpB-P) and mitochondrial (ClpB-M), based on sub-cellular localization (Grover et al., 2013). The transgenic plants for heat tolerance have thus far been attempted with ClpB-C gene. AtHsp100 gene was over-expressed in wild type (WT) A. thaliana plants using CaMV35S promoter (Grover et al., 2013). This experiment was aimed at expressing Hsp100 constitutively, while Hsp100 expression is strictly heat-regulated in vegetative tissues of plants under natural conditions (Singh et al., 2010). The authors of the latter study noted that Hsp100 expression was co-suppressed in most of the transformed plants; only few lines actually showed increased Hsp100 amounts. It further accrued that transgenic A. thaliana plants constitutively-expressing Hsp100 could tolerate sudden shifts to heat stress better than WT plants. In contrast, transformed A. thaliana lines showing co-suppression of the introduced gene had diminished heat tolerance activity. In another instance, AtHsp100 was over-expressed in rice using maize Ubi1 promoter (Grover et al., 2013). The rice transformants showed positive effects on rice tissues, the transgenics expressing Hsp100 showing better growth in the post-heat recovery phase. Further, tobacco plants over-expressing rice Hsp100 protein under CaMV35S promoter survived high temperatures relatively better than the untransformed plants (Grover et al., 2013). Hsp70 and Hsp40 together constitute the bichaperone machine. Hsp70 are highly-conserved chaperones noted in bacteria, plants and animals. These proteins interconnect with several other chaperones in a wide network and are implicated in diverse cellular processes (Wang et al., 2014a,b). Hsp70 are ATP-dependant chaperones containing N-terminal ATPase domain and a variable Cterminal region. ATPase domain performs regulatory role and substrate binding domain binds to substrate proteins. Rice contains 32 proteins in Hsp70 superfamily (24 Hsp70, 8 Hsp110) (Sarkar et al., 2013). Hsp70 protein obtained from halotolerant cynobacterium Aphanothece halophytica was over-expressed in tobacco (Grover et al., 2013). The transgenic plants showed higher thermotolerance during germination and early growth. The tobacco and rice transgenics over-expressed with A. halophytica Hsp70 protein contained higher activity of ascorbate peroxidase (APX) and catalase (Grover et al., 2013). The rice transgenics showed increased activity of Calvin cycle enzymes. The tobacco Hsp70 (Hsp70-1) over-expression prevented fragmentation and degradation of nuclear DNA during heat stress (Grover et al., 2013). The overexpression of Hsp70 from fungus Trichoderma harzianum in A. thaliana resulted in increased level of Naþ/Hþ exchanger1 (SOS1) and APX1 and decreased levels of Hsf and Hsp transcripts in the trans-host (Grover et al., 2013). The transgenics showed increased heat tolerance in this experiment. Over-expression of mitochondrial rice Hsp70 in rice resulted in lesser production of heat induced ROS, higher mitochondrial membrane potential and suppressed programmed cell death (Grover et al., 2013). Constitutive expression of a chrysanthemum Hsp70 in A. thaliana enhanced the tolerance against heat, drought and salinity stresses (Song et al.,

2014). The transgenic plants produced in the latter study showed increase peroxidase activity and proline content and reduced MDA levels under heat stress. Hsp40 (J proteins; also called DnaJ proteins) are large and structurally diverse family of molecular chaperones with a signature J domain. DnaJ proteins, as co-chaperones, work in close proximity with DnaK/Hsp70 members to execute multiple processes during protein homeostasis. T-DNA insertion mutants of A. thaliana for AtDjB1 were found to be thermosensitive having reduced ascorbic acid content and higher levels of ROS after heat stress (Zhou et al., 2012). Transgenic tomato plants with enhanced thermotolerance were generated by over-expression of LeCDJ1, a chloroplast targeted DnaJ protein of tomato (Kong et al., 2014). Transgenic tomato plants showed lesser photoinhibition of PSII and higher APX1 and SOD activities. Hsp20 or sHsps are expressed in maximal amounts under high temperature stress (Sarkar et al., 2009). These are characterized by a C-terminal a-crystalline domain. In concert with Hsp100/Hsp70 and co-chaperones, sHsps prevent cellular protein aggregation and aid in subsequent refolding. sHsps are represented by large families in plants. Rice has 40 a-domain containing genes out of which 23 are sHsps and 17 are aecrystallin domain (ACD) proteins (Sarkar et al., 2009). A. thaliana plants over-expressing Hsp17.5 of Nelumbo nucifera, Hsp17.8 of Rosa chinesis, Hsp22 of Zea mays, Hsp26 of Saccharomyces cerevisiae and Hsp16.45 of Lilium davidii, showed heat tolerance to varied extents (Grover et al., 2013; Mu et al., 2013). Tobacco plants over-expressing Hsp16.9 of Z. mays resulted in increased early seed growth (Grover et al., 2013). This phenotype was associated with enhanced anti-oxidant enzyme activity. Tall fescue (Festuca arundinacea) plants over-expressing Hsp26 of Oryza sativa showed higher PSII activity under heat stress associated with reduced electrolytic leakage and accumulation of thiobarbituric acid reactive substances (Grover et al., 2013). Induction of Hsp gene expression is mediated by the binding of Hsfs at heat shock element (HSE) sequences located in the promoter regions. Hsfs possess an N-terminal DNA binding domain (DBD), an oligomerization domain with a heptad hydrophobic repeat, a short nuclear import motif and an AHA type activation domain in the C-terminus. Plant Hsfs are classified into 3 evolutionary conserved classes, namely, A, B and C. Only the class A members possess the AHA type acidic-activation domain. The class B and C members possess a neutral/basic C-terminal domain. Rice genome contains 26 Hsf proteins (Mittal et al., 2009). As Hsfs regulate expression of diverse down-stream Hsps and a host of other defence-related genes, there is great deal of interest in generating transgenics over-expressing Hsfs. G. max HsfA1 was over-expressed in G. max (Grover et al., 2013). The resulting transgenics showed enhanced heat tolerance through activation of Hsp70. Constitutive expression of HsfA2 in A. thaliana conferred enhanced basal and acquired thermotolerance as well as salt or osmotic stress tolerance (Grover et al., 2013). The over-expression of A. thaliana HsfA3 in A. thaliana caused induction of a large number of heat stress associated genes and showed enhanced heat stress tolerance (Grover et al., 2013). O. sativa Hsf7 over-expression in A. thaliana enhanced expression of certain Hsf target genes, concomitant to increased basal heat tolerance (Grover et al., 2013). Hsf1 from resurrection plant Boea hygrometrica in A. thaliana and Nicotiana tabacum showed enhanced basal and acquired heat tolerance via regulation of genes involved in stress protection and mitotic cell cycle (Grover et al., 2013). The over-expression of HsfA2 from Lilium longiflorum in A. thaliana activated Hsp101, Hsp70, Hsp25.3 and APX2 genes, resulting into heat tolerance of the transgenic plants (Grover et al., 2013). Increased thermotolerance was obtained in transgenic A. thaliana over-expressing wheat HsfA3 (Zhang et al., 2013a,b). Over-expression of tomato HsfA3 in A. thaliana showed increased levels of several Hsp transcripts and


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increased heat tolerance (Li et al., 2013a,b). Transgenic A. thaliana plants over-expressing HsfA1d from Thelluginella salsuginea developed enhanced thermotolerance via induction of AtHsfA1 regulon in the transgenic plants (Higashi et al., 2013). Overexpression of Triticum aestivum HsfA2d, which is expressed mainly in developing seeds, conferred higher tolerance to heat, salinity and drought stresses in A. thaliana in terms of higher survival rate, yield and biomass accumulation (Chauhan et al., 2013). Increased heat resistance was noted in transgenic A. thaliana plants over-expressing a novel class A1 Hsf, LlHsfA1 from L. longiflorum which was found to interact with LlHsfA2 (Gong et al., 2014). 4. Development of heat tolerance through augmentation of overall metabolism Various proteins associated with diverse cellular metabolic activities have been over-expressed in transgenic experiments with the view of enhancing heat tolerance. These include proteins implicated in metabolism of amino acids and derivatives, protein biosynthesis, photosynthetic activity, redox homeostasis and hormonal regulation. L-aspartate-a-decarboxylase has role in b-alanine biosynthesis. E. coli gene encoding for this protein when overexpressed in tobacco resulted in high heat tolerance (Grover et al., 2013). Avena sativa gene for arginine decarboxylase enzyme (involved in polyamine biosynthesis) resulted in enhanced thermotolerance in Saccharomyces melongena (Grover et al., 2013). Over-expression of S. cerevisiae gene encoding for S-adenosyl-Lmethionine decarboxylase (SAMDC) enzyme (involved in polyamine biosynthesis) increased the high temperature tolerance of transgenic Solanum lycopersicum (Grover et al., 2013). SAMDC gene from Homo sapiens also was effective in this context (Grover et al., 2013). Higher endogenous spermine levels engineered by overexpressing spermine synthase gene in A. thaliana correlated with increased tolerance to heat stress (Sagor et al., 2013). Protein biosynthesis process involves a battery of factors controlling initiation, elongation and termination processes. R. chinesis gene encoding for translation initiation factor resulted in high heat stress tolerance when over-expressed in A. thaliana (Grover et al., 2013). Z. mays gene encoding for elongation factor resulted in high heat stress tolerance when over-expressed in T. aestivum (Grover et al., 2013). Apart from Hsp chaperones discussed in the preceding account, diverse other chaperone proteins have been implicated in heat stress response. Over-expression of AtFKBP62 gene in A. thaliana resulted in increased heat stress tolerance (Grover et al., 2013). Likewise, Cajanas cajan gene encoding for cyclophilin chaperone when over-expressed in A. thaliana resulted in heat tolerance (Grover et al., 2013). A. thaliana gene for thioredoxin-like protein (a foldase and holdase chaperone) resulted in increased heat tolerance when over-expressed in A. thaliana (Grover et al., 2013). ROS scavenging pathways are of prime importance in plant stress responses. A. thaliana gene encoding for nucleotide diphosphate kinase (ROS scavenging enzyme) was over-expressed in Solanum tuberosum, resulting in increased heat tolerance (Grover et al., 2013). S. lycopersicum gene for GDP-mannose pyrophosphorylase (GMPase; ROS scavenging enzyme) when over-expressed in N. tabacum resulted in high heat tolerance as a result of increased accumulation of ascorbic acid which acted as a strong reductant in transgenic plants (Grover et al., 2013). Antisensing of tobacco endogenous GMPase led to decreased ascorbic acid content, chloroplastic SOD and APX activities, Pn, Fv/Fm, lower germination rate and increased ROS accumulation under heat stress (Wang et al., 2012a,b). Antisense transgenics had smaller leaves and earlier onset of flowering. O. sativa gene for chloroplast protein-enhancing stress tolerance resulted in high heat tolerance when over-

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expressed in A. thaliana (Grover et al., 2013). Further, A. thaliana gene for cytokinin oxidase/dehydrogenase (cytokinin deactivating enzyme) when ectopically expressed in N. tabacum, resulted in high et al., 2013). General stress related proheat tolerance (Mackova teins have been ectopically over-expressed in some cases. Xerophyta viscosa gene encoding for stress associated protein 1 (SAP1; a cell membrane binding protein) was over-expressed in A. thaliana, resulting in high heat tolerance (Grover et al., 2013). Populus tremula gene encoding for stable protein provided increased heat tolerance when over-expressed in A. thaliana (Grover et al., 2013). A. thaliana gene for SAP5 when over-expressed in Gossypium hirsutum resulted in enhanced heat tolerance (Grover et al., 2013). The general mode of action of SAPs under abiotic stresses has not been defined yet. A. thaliana SAP5 was suggested to positively regulate salt and osmotic stress tolerance through its E3 ubiquitin ligase activity (Kang et al., 2011). Over-expression of Medicago truncatula SAP1 in N. tabacum exhibited greater biomass, primary root length and leaf development under heat and cold stresses along with enhanced osmotic and salt stress tolerance (Charrier et al., 2013). Interestingly, proline content was unaltered under salt and osmotic stresses in transgenic plants, in contrast to wild type, suggesting that MtSAP1 mediated stress tolerance was independent of free proline content. Diverse other proteins have been employed in transgenic experiments like Agrobacterium rhizogenes gene encoding for bglucosidase (involved in tumor formation and hairy root disease) over-expressed in Rubia cordifolia, C. cajan gene for hybrid prolinerich protein (cell wall protein) over-expressed in A. thaliana, Malus domestica gene encoding for vacuolar proton translocating inorganic pyrophosphatase (role in ion transport) over-expressed in M. domestica, Z. mays gene encoding for acetyl cholinesterase (involved in xenobiotic hydrolysis) over-expressed in N. tabacum and A. thaliana gene for CYP710A1 (involved in conversion of sitosterol into stigmasterol) over-expressed in A. thaliana (Grover et al., 2013; Senthil-Kumar et al., 2013). Annexin protein from N. nucifera led to enhanced heat stress tolerance in transgenic A. thaliana seeds upon ectopic expression accompanied with reduced lipid peroxidation (Chu et al., 2012). In all above and in other cases shown in Table 2, heat tolerance responses, quantified using a plethora of cellular parameters, were enhanced in transgenic progenies. 5. The road ahead for future research In experiments carried out thus far, extension of laboratory based success to field conditions has not been realized. It is thus likely that the individual genes over-expressed in transgenic experiments have not resulted in sufficient level of stress tolerance that is reflected throughout plant growth and development. The important facet of heat stress tolerance is that it is a polygenic trait compounded by complex genotype-environment interactions. The gene transfers carried out by over-expressing Hsps and diverse metabolic proteins may not affect the gene expression comprehensively. It is essential to elucidate the ‘master-regulator’ stress genes in future years. Hsfs acting at upstream level can possibly affect large number of effector genes. So far, the master-regulator Hsf genes have been claimed only in tomato and A. thaliana (Grover et al., 2013). However, the over-expression of Hsfs and analysis of the down-stream Hsp genes have been followed to a limited extent. Increasing evidences suggest that heat shock response pathways activated by Hsfs are regulated by feedback loops operating via specific interactions between Hsfs and Hsps (Fragkostefanakis et al., 2014). It is imperative to reveal such regulatory circuits including the identification and characterization of additional key players wiring these and possibly acting as nodes


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and hubs for stress signaling cross-talk. The upstream regulators of Hsf gene expression may be useful candidates for modulating heat resistance through coalition of downstream signaling pathways. The characterization of plant thermosensor mechanisms is important as thermosensor proteins can be used as novel masterregulator genes for engineering thermotolerance. The ‘systems biology’ approach for integration of the ‘omics’ data in conjunction with mathematical modeling may prove more effective in designing high heat tolerance. The perils of high temperature stress under field conditions are added on by drought, salinity, ultra violet radiation, pathogens etc. Recent studies indicate that combinatorial action of multiple stresses illicit unique genic responses as compared to high temperature stress alone (Grover et al., 2013; Rasmussen et al., 2013; Sewelam et al., 2014). There is strong need to identify genes crucial in responses against combination of stresses. For effective selection of transgenics with high heat tolerance in field conditions, efficient and uniform phenotyping protocols need to be developed that can mimic the complex field stress regimes. The constitutive expression of stress tolerance related genes often negatively affects the growth and yield of transgenic plants. Spatio-temporally controlled expression of transgenes must be ascertained by the use of tissue-, stage- and stress-inducible promoters in future course of experiments. Future research with suitably tailored expression tools driving masterregulator genes like thermosensors and Hsfs would enable production of novel and superior heat tolerant transgenic crops. Contributions MHS and MHA-W contributed on details on molecular biology of heat stress response of plants. DL, AD and AG contributed on genetic transformation of heat stress resistance. This manuscript is a combined effort of all the authors. Uncited reference Stocker et al., 2013. Acknowledgments DL is thankful to Council of Scientific and Industrial Research, Government of India and University Teaching Assistant fellowship, University of Delhi for the research fellowship award. MHS and MHA-W thank project funding from National Plan for Science and Technology Program, Saudi Arabia. AG gratefully acknowledges Visiting Professorship of King Saud University, Saudi Arabia. References Barah, P., Jayavelu, N.D., Mundy, J., Bones, A.M., 2013. Genome scale transcriptional response diversity among ten ecotypes of Arabidopsis thaliana during heat stress. Front. Plant Sci. 4, 1e10. Bita, C.E., Gerats, T., 2013. Plant tolerance to high temperature in a changing environment: scientific fundamentals and production of heat stress-tolerant crops. Front. Plant Sci. 4, 1e18. Bita, C.E., Zenoni, S., Vriezen, W.H., Mariani, C., Pezzoti, M., Gerats, T., 2011. Temperature stress differentially modulates transcription in meiotic anthers of heat-tolerant and heat-sensitive tomato plants. BMC Genomics 12, 384. Boden, S.A., Kavanov a, M., Finnegan, E.J., Wigge, P.A., 2013. Thermal stress effects on grain yield in Brachypodium distachyon occur via H2A.Z-nucleosomes. Genome Biol. 14, R65. Chae, H.B., et al., 2013. Thioredoxin reductase type C (NTRC) orchestrates enhanced thermotolerance to Arabidopsis by its redox-dependent holdase chaperone function. Mol. Plant 6, 323e336. vre, E., Limami, A.M., Planchet, E., 2013. Medicago truncatula stress Charrier, A., Lelie associated protein 1 gene (MtSAP1) overexpression confers tolerance to abiotic stress and impacts proline accumulation in transgenic tobacco. J. Plant Physiol. 170, 874e877. Chaturvedi, P., Ischebeck, T., Egelhofer, V., Lichtscheidl, I., Weckwerth, W., 2013. Cell-specific analysis of the tomato pollen proteome from pollen mother cell to

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Corrigendum: Status and market potential of transgenic biofortified crops.

Mixotrophic cultivation of microalgae for biodiesel production: status and prospects.

Model biases in rice phenology under warmer climates.

Production of virus resistant and insect tolerant transgenic tobacco plants.

low-temperature cultivation.

MAGIC populations in crops: current status and future prospects.

Iron-biofortified staple food crops for improving iron status: a review of the current evidence.

Network Candidate Genes in Breeding for Drought Tolerant Crops.

[Current status of the High 5s Project].

Proteomics of Important Food Crops in the Asia Oceania Region: Current Status and Future Perspectives.

Evaluation of the agronomic performance of atrazine-tolerant transgenic japonica rice parental lines for utilization in hybrid seed production.

The Regulatory Status of Genome-edited Crops.

High-temperature cultivation of recombinant Pichia pastoris increases endoplasmic reticulum stress and decreases production of human interleukin-10.

Current status of radical prostatectomy for high-risk prostate cancer.

Optimizing pyramided transgenic Bt crops for sustainable pest management.

Human health and transgenic crops symposium introduction.

A high-temperature tolerant species in clade 9 of the genus Phytophthora: P. hydrogena sp. nov.

Outcrossing and hybridization in wild and cultivated foxtail millets: consequences for the release of transgenic crops.

Basic loci in cultivation of certain crops in the past and modern times.

Biotechnological production of muconic acid: current status and future prospects.

Production of arabitol by yeasts: current status and future prospects.

High-Intensity Focused Ultrasound: Current Status for Image-Guided Therapy.

Response surface optimization for xylanase with high volumetric productivity by indigenous alkali tolerant Aspergillus candidus under submerged cultivation.

Laccase applications in biofuels production: current status and future prospects.