Jianchang Yang*,Wenqian Miao,Jing Chen

Jiangsu Key Laboratory of Crop Genetics and Physiology/Co-Innovation Center for Modern Production Technology of Grain Crops,College of Agriculture,Yangzhou University,Yangzhou 225009,Jiangsu,China

Keywords:Brassinosteroids High temperature stress Jasmonates Rice Thermotolerance

ABSTRACT High temperature(HT)stress has become one of the most detrimental stresses in crop production among constantly changing environmental factors.Exploiting approaches to enhance crop thermotolerance would have great significance in assuaging adverse effects of HT stress on crop growth and development.As jasmonates (JAs) and brassinosteroids (BRs) are novel phytohormones and play important roles in responses to biotic and abiotic stresses and in a wide range of plant developmental processes,this paper reviewed the roles and mechanisms of JAs and BRs in mitigating HT stress,with focus on rice(Oryza sativa L.) subjected to HT stress during anthesis.It is demonstrated that JAs alleviate spikelet-opening impairment and BRs ameliorate pistil fertilization ability under HT stress during anthesis of rice,although there are controversial observations.Activating the defense system,enhancing osmotic regulation,protecting photosynthesis,and interacting with other phytohormones,especially with ethylene and abscisic acid,are main physiological mechanisms by which JAs or BRs attenuate HT stress to plants.Elevating levels of JAs or BRs in plants could be considered as an important approach to enhance crop thermotolerance through breeding new varieties.Using JAs or BRs as chemical regulators and adopting proper water and nitrogen management practices could reduce the harm of HT stress to rice.Further research is needed to elucidate the roles of JAs and BRs in different plant tissues in responses to HT stress under different genetic backgrounds and environments,reveal the molecular mechanism underlying JAs and BRs mediating HT stress,understand the cross-talk between phytohormones in modulating HT stress,and establish integrated crop management to minimize the hazard of HT stress in rice production.

Contents

1.Introduction .........................................................................................................978

2.Roles of JAs in mediating the effects of HT stress ...........................................................................978

3.Roles of BRs in modulating the effects of HT stress..........................................................................979

4.Interaction between JAs,BRs,ethylene and ABA ............................................................................979

5.Cultivation management techniques to cope with HT stress ..................................................................980

6.Concluding remarks...................................................................................................981

6.1.The roles of JAs and BRs at different conditions .......................................................................981

6.2.The molecular mechanism underlying JAs and BRs in responses to HT stress ...............................................981

6.3.The cross-talk between phytohormones in modulating HT stress.........................................................981

6.4.Integrated crop management to reduce HT stress .....................................................................982

Declaration of competing interest .......................................................................................982

Acknowledgments ....................................................................................................982

References ..........................................................................................................982

1.Introduction

With the increase in global greenhouse effect and changes in climate,high temperature(HT) stress has become one of the most detrimental stresses among constantly changing environmental factors[1–3].It has been predicted that the global mean temperature will rise by 4–5 °C till the end of this century [4],while rice yield could be decreased approximately by 10% for each 1 °C increase in the minimum temperature during the growing season[5].The flowering or anthesis stage in rice plants is most vulnerable to HT stress [6–8].For a conventional male fertile rice cultivar or a line,HT stress (≥35 °C) during anthesis usually causes failure in anther dehiscence,pollination,and germination of pollen grains,leading to a low seed-setting rate and a low grain yield [8–10].In contrast,for a photothermo-sensitive genetic male-sterile(PTSGMS) rice line that shows total male sterility when temperature is above 23 °C or 24 °C [11–14],HT stress during anthesis mainly impairs spikelet-opening and reduces pistil activity,and consequently,seriously decreases grain yield of a hybrid produced by the line crossed with a restorer line [15–18].

Usually,HT stress during anthesis in cereal crops affects activities of the key enzymes involved in the metabolism of nucleic acids,proteins and sugars,such as nuclease,protease,invertase,and amylase in microspore and anthers[10,19–21],increases reactive oxygen species(ROS),and enhances or inhibits the expression of genes encoding antioxidant system (AOS) and some proteins,especially heat shock proteins (HSPs),in anthers and tapetum,which result in anther abnormality,floret-or spikelet-opening impairment,and pollen or spikelet sterility [22–26].Meanwhile,the reduction in photosynthetic rate and the inhibition in photosynthetic efficiency of photosystem II (PSII) and photochemical activity associated with photosystem I (PSI) caused by HT stress aggravate spikelet sterility and seed yield loss [27–29].

It is generally believed that phytohormones are signaling molecules and play vital roles not only in regulating plant growth and development,but also in mediating the physiological processes responding to a variety of biotic and abiotic stresses including HT stress[30–34].Among them,jasmonates(JAs)and brassinosteroids(BRs) have attracted great attention because they have been observed to minimize the damage of HT stress to plants in recent studies [15–18,35–38],although there are controversial observations[39–41].Understanding the roles of JAs and BRs in responses to HT stress would have great significance to elucidate the mechanism in which plants adapt to HT stress and explore new approaches alleviating the stress to crops.Therefore,the paper reviewed roles and mechanism of BRs and BRs in mitigating HT stress in plants,with focus on PTSGMS rice lines subjected to HT stress during anthesis.The interactions of JAs and BRs with other hormones,such as ethylene and abscisic acid (ABA),and the crop management to enhance rice thermotolerance were also discussed.

2.Roles of JAs in mediating the effects of HT stress

Jasmonates,including jasmonic acid(JA)and methyl jasmonate(MeJA),are a class of polyunsaturated fatty acid-derived phytohormones and ubiquitous in higher plant species [42,43].They play crucial roles in regulating plant growth and development including floret development,lodicule swelling and withering,anther dehiscence,filament elongation,and pollen maturation [44–46].It is observed that JA deficiency could inhibit lodicule expansion,leading to scattered floret opening time in a cytoplasmic male sterile(CMS) rice line [47].When a CMS rice line was treated with MeJA before anthesis,lodicule expansion was substantially induced,the number of opening florets was almost doubled,and the percentage of filled spikelets of the female parent was increased by 400%,in comparison with those of a non-MeJA-treated CMS line[48].Investigations on delayed dehiscence mutants clarify that JAs are essential for anther dehiscence and pollen maturation inArabidopsis[49,50].

There are many reports showing that JAs play positive roles in response to abiotic stresses including salt,drought,flooding,heavy metals,micronutrient toxicity,and low temperature [51–55].It is reported that JAs could confer thermotolerance by enhancing the expression of genes in the JA biosynthesis and signal pathways inArabidopsis[35,56].Recent studies have shown that a PTSGMS rice line with higher JA and MeJA contents in the lodicules under HT stress during anthesis exhibited a higher opened-spikelet rate and also showed a higher fertilized-spikelet rate and a higher seed yield when the line was crossed with a restorer line,and both JA and MeJA contents were very significantly and positively correlated with fertilized-spikelet rate [15,16,18].Furthermore,increases in JAs contents in lodicules by applying JA or MeJA significantly increased opened-spikelet rate of both PTSGMS and restorer lines subjected to HT stress during anthesis [18].Importantly,application of JA or MeJA was only observed to promote spikelet-opening of rice,which was reflected by an increase in spikelet-opening rate under the HT stress (Table 1).Applying zeatin riboside (ZR,a kind of cytokinins),indole-3-acetic acid (IAA),gibberellin acid-3 (GA3),24-epibrassinolide (24-EBL,a form of BRs),or ABA at low concentration(10 μmol L-1)had no significant effect on spikelet-opening rate.Application of ABA at high concentration(50 μmol L-1)or 1-aminocyloprane-1-carboxylic acid(ACC,the precursor of ethylene synthesis) significantly reduced the spikelet-opening rate (Table 1).These results indicate that,among phytohormones,JAs may play a unique role in alleviating spikeletopening impairment caused by HT stress during anthesis in rice.

The physiological mechanism by which JAs ameliorate tolerance to abiotic stresses especially to HT stress during anthesis could be mainly explained in two aspects.Firstly,JAs could activate the defense system of plants,mainly via enhancing activities of antioxidative enzymes including catalase (CAT),superoxide dismutase(SOD),guaiacol peroxidase,glutathione peroxidase and increasing other defensive compounds such as ascorbic acid (AsA) and HSPs,thereby suppressing ROS generation and reducing programmed cell death under stresses [16,18,57–59].Secondly,JAs could enhanceosmotic regulation by increasing the synthesis of some asmoregulators,such as proline,phenolic compounds,and soluble carbohydrates,and accordingly,reduce the harm of stresses to plant organs,stimulate plant cell expansion,and promote the spikeletopening of rice[16,18,60–62].It is also possible that JAs may interact with other phytohormones to mediate the effect of HT stress on plant growth and development,which was discussed below.

It should be noteworthy that there is a controversial observation that JA level is decreased and JA biosynthetic genes are down-regulated in rice seedling leaves under heat stress [39].The levels of both JA and MeJA in rice lodicules,however,have been observed to be increased at the first day and decreased on the third day of HT treatments during anthesis[16,18].These studies imply that the decrease or increase in JAs levels in plants may depend on plant organs and growth stages,the treatment time,and the duration of HT stress.

3.Roles of BRs in modulating the effects of HT stress

Brassinosteroids are a unique class of naturally occurring plant polyhydroxylated steroid hormones and comprised of brassinolide(BL),castasterone,and their various derivatives,such as 24-epibrassinolide (24-EBL),homobrassinolide (28-HBL),and 24-epicastasterone [64,65].They play vital roles in plant growth and development,including seed germination,spikelet differentiation,pollen tube elongation,and flower and fruit production by regulating physiological and molecular processes,such as cell division,nucleic acid and protein biosynthesis,gene expression,and photosynthesis[66–69].These phytohormones can also confer resistance to biotic and abiotic stresses including drought,flood,salinity,extreme high and low temperatures,heavy metal toxicity,ozone,pests,and pathogens [41,70–72].It is found that loss-of-function mutations in either BR biosynthetic or signaling genes exhibit sensitive characteristics to heat stress compared to wild type inArabidopsis thaliana[37].Brassinosteroids have been observed to attenuate the detrimental effects of HT stress on leaf photosynthesis,seed germination,and yield quantity and quality of crops [73–77].Recent work of Chen et al.[17] has demonstrated that BRs can mediate the effect of HT stress during anthesis on pistil fertilization ability and alleviate the harm of HT stress to the pistil fertility in PTSGMS rice lines.All these observations suggest that BRs may act as immunomodulators to protect plants from injuries of HT stress.

The mechanism underlying BRs mitigating adverse effects of HT stress on plant growth and development has been revealed either physiologically or molecularly[69–76].It is proposed that BR perception takes place in the cell surface by BR receptors,leading to a variety of phosphorylation events to activate the central transcription factor BRASSINAZOLE-RESISTANT1 (BZR1) that controls the transcription of BR-responsive genes in the nucleus,which functions as critical signal to ameliorate tolerance to detrimental environmental conditions,especially to HT stress [72,78,79].There are reports showing that BRs could increase the production of HSPs under HT stress,thereby protecting proteins against irreversible heat-induced damage by preventing denaturation and facilitating the refolding of damaged proteins [80–83].Brassinosteroids have been observed to maintain redox homeostasis under HT stress by enhancing activities of enzymes involved in the ascorbate–glutathione (AsA-GSH) cycle including CAT,SOD,ascorbate peroxidase,glutathione reductase,dehydroascorbate reductase,and monodehydroascorbate reductase and the expression of genes encoding these enzymes [76,84–88].A major role of BRs in responding to HT stress is reported to protect plant photosynthesis through improving the photosynthetic pigment contents,carboxylation rate of Rubisco,photochemical activity of PSI,and energy charge [29,89–91].The cross-talk of BRs with other phytohormones may also contribute to minimizing HT stress [91–93].

Notably,there is a report showing that BR-deficient and BRsignalling mutants exhibited higher tolerance to high temperatures than the wild types at the seedling stage of barley [93].SixBZRgenes in maize seedlings have been observed to be downregulated under HT stress [94].It is argued that BRs may not produce HSPs to augment thermotolerance because BR-deficient mutants inArabidopsis thalianastill highly expressed HSPs during HT stress[95].These observations suggest that the roles and mechanisms of BRs in mediating the effects of HT stress on plant growth and development greatly vary depending on the plant species,developmental stages,and environmental conditions.

4.Interaction between JAs,BRs,ethylene and ABA

It has been proposed that JAs,BRs,ethylene,and ABA are major phytohormones that can act either synergistically or antagonistically to mediate biotic and abiotic stresses [91–93,96–98].Jasmonic acid has been observed to antagonize ethylene in regulating HT stress [35].Arabidopsismutants showing either JA biosynthesis or JA signaling are sensitive to heat stress,while the ethylene mutantethylene insensitive2-1(ein2-1) conferred greater thermotolerance asEIN2-mediated pathway negatively regulates thermotolerance [56,99].On other hand,JAs have been observed to induce ethylene biosynthesis by enhancing activities of the ethylene making enzymes in tomato [100].The JA-ethylene interaction has been proposed to be linked mainly through the ethylene-activated transcription factor EIN3 and its close homolog EIN3-Like1 (EIL1) [101–103].A possible antagonism between BRs and ethylene has been speculated by Chen et al.[17],which is supported by three observations:(1) a PTSGMS rice line with higher BRs(24-EBL and 28-HBL)contents under HT stress during anthesis exhibited lower ACC content in the pistils;(2) contents of 24-EBL and 28-HBLsignificantly and positively,whereas ACC content negatively,correlated with AsA content and catalase activity in pistils of PTSGMS lines;and(3)application 24-EBL or 28-HBL to the panicles of PTSGMS lines significantly decreased,whereas applying brassinazole (an inhibitor of BR biosynthesis) significantly increased,ACC content in the pistils.However,direct evidences on the cross-talk between BRs and ethylene are still lacking.

A synergistic action between JA and ABA under HT stress has been suggested,which is supported by the fact that foliar spray of JA increased ABA content in plant cells,thereby stimulating stomata closing and preserving water content [104–106].However,an antagonism between JA and ABA was observed in rice response to nematode attack and saline stress [107,108].The antagonistic interaction between BRs and ABA has also been suggested in plant response to HT stress[109].It is observed that high endogenous levels of ABA could suppress BR-mediated responses to heat stress inArabidopsis,and application of BRs to an ABAdeficient mutant exhibited pronounced effects due to higher accumulation of HSP 90 (109).There is a speculation that ABA may minimize the role of BRs in heat stress and this interaction might involve the down-regulated expression ofBZRgenes in maize[94].The work of Kurepin et al.[110] has demonstrated,however,that application of BL or 24-EBL toBrassica napusleaves could substantially increase endogenous ABA concentration and enhance tolerance to HT stress.It is argued that BRs may play a role in maintaining the ABA-ABA glucosylester balance to alleviate the effect of high and low temperatures on barley [92].These results indicate that BRs may interact with ABA not only antagonistically but also synergistically on plant responses to HT stress.

Several studies have shown an antagonistic interaction between JAs and BRs on regulating plant growth [111–114].For example,MeJA is observed to inhibit lamina joint inclination by repressing BR biosynthesis and signaling in rice leaves [113],and application of MeJA substantially represses the expression of BR biosynthesisrelated genes,decreases endogenous BRs levels,and activates the JA-dependent rice innate immunity against root-knot nematodes of rice roots [113,114].However,it is reported that application of vitamin E (Ve)+vitamin C (Vc)+MeJA+BR exhibited the best to assuage the adverse effect of HT stress during anthesis on the pollen fertility and germination of rice among the four treatments (other three treatments:BR+triazoles+MeJA;Ve+Vc;and MeJA)[115].A PTSGMS rice line with higher BRs contents also shows higher JA or MeJA contents under HT stress,and they are significantly correlated [16–18].Application of MeJA or 24-EBL to heat stressed PTSGMS rice lines during anthesis significantly enhanced the expression of genes related to JAs and BRs biosynthesis or signaling in the pistils,whereas the results were reversed when neomycin(an inhibitor of JA-mediated reactions) or brassinazole was applied(Table 2).Furthermore,simultaneously application of MeJA and 24-EBL more enhanced pistil vigor of PTSGMS rice lines which was reflected by a higher AsA content and a lower H2O2content in the pistil than application of MeJA or 24-EBL alone(Table 3).Therefore we speculate that there may exist synergistic interplay between JAs and BRs for regulating rice responses to HT stress.More convinced evidences,however,remain to be provided to back such a speculation.

Table 2 Effects of applying chemical regulators on the expression of genes related to JAs and BRs biosynthesis or signaling in rice pistils subjected to high temperature stress during anthesis.

It is worthy of attention that salicylic acid(SA),another kind of novel phytohormones,has been suggested to promote basal thermotolerance and induce membrane thermoprotection[116].However,an antagonistic interaction between JA and SA has been observed to respond to HT stress in signaling pathways[117,118].Brassinosteroids are also proposed to interact antagonistically with SA to regulate HT stress,which is evidenced by the fact that endogenous contents of SA inArabidopsiswere decreased when plants were treated with BR under HT stress[37].It is argued that JAs and BRs can enhance both basal thermotolerance and heat stress acclimation,whereas SA has no function in acquiring heat stress acclimation [119].

5.Cultivation management techniques to cope with HT stress

Use of heat-tolerant varieties is an effective way to enhance crop thermotolernce[120].Moreover,adoptions of proper cultivation management techniques could either aviod or alleviate HT stress in crop production [120–123].Among them,application of nitrogen (N) fertilizer and irrigation are considerted as two most important crop management practices to reduce the harm of HT stress to rice [124–128].When compared with a lower N rate or no N application,a higher N rate or application of N granular fertilizer significantly increased seed-setting percentage,1000-grain weight,and grain yield of rice under the HT stress during anthesis,mainly via increases in root oxidation activity and leaf photosynthesis,enhancement in activities of the enzymes involved in N metbolism in leaves and sucrose-starch conversion in the grain,and the amelioration in AOS [125,126,129].The optium N rate to enhance rice termotolerance may vary with soil fertility,N levels in plants,rice varieties,and growth stages.It is observed that application of N fertilizer could significantly attenuate the effect of HT stress on spikelet-opening and pistil activity of PTSGMS rice lines when N content in plants is lower than 2.3%–2.4%during anthesis,and the effectiveness of N application could be decreased when N content in plant is higher than the value [63].

A proper irrigation regime is an effective real-time cultivation measure to enhance rice thermotolerance[127,128].Usually,when HT stress happens at the heading and flowering stage of rice,flood water or deep water is applied to paddy field to reduce the adverse effect of HT stress on plants by increasing humidity and evaporation,and thereby cooling canopy [128,130–133].However,There are observations that the injury of HT stress to rice is closely associated with canopy humidity,and the higher the relative humidity,the more serious the injury under the same HT condition[134,135].It is argued that although irrigation with deep water could reduce the canopy temperature,it also increases the canopy humidity [134,135].Some researchers [134,135] have observed that,under HT stress during anthesis of rice and when compared with continuously flooded irrigation,an alternate wetting and moderate soil drying regime (plants were re-watered when soil water potential reached to -15 kPa) significantly increased seedsetting rate,1000-grain weight,and grain yield.The better yield performance under such an irrigation regime was mainly attributed to the reduction of relative humidity in the panicle canopy,increased transpiration rate of leaves,decreased reactive oxygen production rate,and elevated concentrations of cytokinins in leaves and spermidine and spermine in the grain [134,135].There is also a report showing that mist spray is an effective measure toreduce the injury of HT stress during anthesis of rice,due mainly to an increase in latent heat flux and a decrease in sensible heat flux in the rice field [127].

Apart from application of N fertilizer and irrigation,adjusting sowing date of rice is another cultivation practice to avoid or reduce the effect of HT stress during the reproductive period especially during anthesis [120,136–138].It is estimated that delaying sowing date of rice to achieve mean daily temperature at 24.9–26.4 °C during the first 20 days after heading can be an effective strategy to deal with the climate change especially HT stress in central China,and thereby improving grain yield and resource use efficiency [137].

6.Concluding remarks

High temperature stress presents an alarming risk to food and nutritional security worldwide [59].The finding that JAs and BRs can mitigate the adverse effect of heat stress on plants provides promising approaches to cope with such a risk.Firstly,crop varieties with strong thermotolerance could be bred by using classical breeding methods and/or genetic engineering technology to enhance JAs or BRs biosynthesis.The approach is possible because variations in endogenous levels of JAs or BRs have been observed among PTSGMS rice lines,and a line with higher levels of JAs or BRs exhibits stronger tolerance to HT stress [16–18].Secondly,either JAs or BRs could be used as chemical regulators to ameliorate crop growth and development under HT stress.Thirdly,adoption of proper water and nitrogen (N) management practices may elevate JAs or BRs levels in plants thereby attenuating the hazard of HT stress to crops,which is evident by the observations that proper application of N fertilizer and water management enhance BRs biosynthesis in rice plants,leading to alleviation in drought stress during spikelet development [139,140].Activating the defense system,enhancing osmotic regulation,protecting photosynthesis,and interacting with other phytohormones,especially with ethylene,ABA,are main physiological mechanisms by which JAs or BRs assuage HT stress to plants(Fig.1).However,studies on JAs or BRs in response to HT stress are rather scarce,and the knowledge to understand roles and mechanisms of JAs or BRs in modulating HT stress to crops is very limited.Therefore,further researches are needed,and may focus on four aspects.

6.1.The roles of JAs and BRs at different conditions

Roles of JAs and BRs in regulating plant growth and development may vary with genetic backgrounds and plant growth conditions.For instance,MeJA has been observed to function as an inducer of spikelet-opening of rice[47,48],and application of MeJA to CMS rice lines before anthesis at normal temperature or to PTSGMS rice lines during anthesis under HT stress substantially increased both spikelet-opening rate and filled-spikelet percentage of the female parents [16,48].By contrast,application of MeJA to the panicles of inbred rice at 0900 during anthesis markedly decreased seed-setting rate resulted from an increase in spikelet sterility due to MeJA-induced early opening of flowers that were expected to open on the next day under non HT stress conditions[141].In addition,the increase or decrease in JAs levels in plants under heat stress varies with rice varieties or lines[16,18,63,130].Therefore,it is necessary to verify the roles of both JAs and BRs in responding to HT stress under different conditions including genetic backgrounds,plant growth stages,plant tissues,and environmental factors.

6.2.The molecular mechanism underlying JAs and BRs in responses to HT stress

So far,explanation on the mechanism underlying JAs and BRs in responses of crop plants to HT stress is mainly based on the observations of endogenous hormonal levels and other physiological parameters and the correlations between the hormonal levels and the determined parameters,which could not verify the cause-and-effect relationship between them[92,98].More investigations are needed to elucidate the molecular mechanism by which JAs and BRs mediate effects of HT stress on plants by using mutants and/or transgenic plants of crops with attenuated capacity to respond to,or synthesize,these two hormones.

6.3.The cross-talk between phytohormones in modulating HT stress

Current studies on the interaction between JAs and BRs or cross-talk of the two regulators with other phytohormones in mediating HT stress are mostly conducted under experiments with exogenous application of these hormones.Evidence for both synergistic and antagonistic interaction between phytohormones in the response could be raised because of the differences in genetic backgrounds and growth conditions of plants under such experiments [36,95].It is presumed that all phytohormones can interact with each other,thereby not only participating in plant developmental processes but also responding to abiotic stresses [99].Owing to the extreme complexity of the interaction between or among phytohormones in the responses,the knowledge on interplay of JAs or BRs with other hormones in crop plants in responses to abiotic stresses,especially to HT stress,is largely unknown or even confusing [32,142].Further researches are necessary to understand the cross-talk between phytohormones,especially between JAs or BRs with other phytohormones,in mediating HT stress from spatiotemporal responses (plant tissues and growth stages),genetic plasticity and its adaptability to environments,the mechanistic basis of tolerance to HT stress,and regulatory effects of crop management on the cross-talk.

6.4.Integrated crop management to reduce HT stress

There are several cultivation practices that could alleviate HT stress to rice at different degrees [124–128,136–138].However,it is integrated crop management,rather than a single cultivation practice,can minimize the hazard of HT stress in the crop production[120–122,143].Further research is necessary to establish integrated crop management,including the integration of selecting thermo-tolerant varieties,adjusting sowing date,accurately applying N and water,and using JAs or BRs as chemical regulators,to enhance rice thermotolerance,and thereby effectively reducing HT stress,and to elucidate the physiological and ecological mechanism underlying the thermotolerance enhancement under such an integrated crop management system.

CRediT authorship contribution statement

Jianchang Yang,Wenqian Miao,and Jing Chen:performed the research and wrote the paper.All authors reviewed the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31771710,32071943),the National Key Research and Development Program of China (2018YFD0300800),and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD-1).