Regulation and physiological function of proteins for heat tolerance in cowpea (Vigna unguiculata) genotypes under controlled and field conditions.

The expression of heat shock proteins is considered a central adaptive mechanism to heat stress. This study investigated the expression of heat shock proteins (HSPs) and other stress-protective proteins against heat stress in cowpea genotypes under field (IT-96D-610 and IT-16) and controlled (IT-96D-610) conditions. Heat stress response analysis of proteins at 72 h in the controlled environment showed 270 differentially regulated proteins identified using label-free quantitative proteomics in IT-96D-610 plants. These plants expressed HSPs and chaperones [BAG family molecular chaperone 6 (BAG6), Multiprotein bridging factor1c (MBF1C) and cold shock domain protein 1 (CSDP1) in the controlled environment]. However, IT-96D-610 plants expressed a wider variety of small HSPs and more HSPs in the field. IT-96D-610 plants also responded to heat stress by exclusively expressing chaperones [DnaJ chaperones, universal stress protein and heat shock binding protein (HSBP)] and non-HSP proteins (Deg1, EGY3, ROS protective proteins, temperature-induced lipocalin and succinic dehydrogenase). Photosynthesis recovery and induction of proteins related to photosynthesis were better in IT-96D-610 because of the concurrent induction of heat stress response proteins for chaperone functions, protein degradation for repair and ROS scavenging proteins and PSII operating efficiency (Fq'/Fm') than IT-16. This study contributes to identification of thermotolerance mechanisms in cowpea that can be useful in knowledge-based crop improvement.

Proteome changes and associated physiological roles in chickpea (Cicer arietinum) tolerance to heat stress under field conditions.

Interrogative proteome analyses are used to identify and quantify the expression of proteins involved in heat tolerance and to identify associated physiological processes in heat-stressed plants. The objectives of the study were to identify and quantify the expression of proteins involved in heat tolerance and to identify associated physiological processes in chickpea (Cicer arietinum L.) heat-tolerant (Acc#7) and sensitive genotype (Acc#8) from a field study. Proteomic and gene ontological analyses showed an upregulation in proteins related to protein synthesis, intracellular traffic, defence and transport in the heat-tolerant genotype compared to the susceptible one at the warmer site. Results from KEGG analyses indicate the involvement of probable sucrose-phosphate synthase (EC 2.4.1.14) and sucrose-phosphate phosphatase (EC 3.1.3.24) proteins, that were upregulated in the heat-tolerant genotype at the warmer site, in the starch and sucrose pathway. The presence of these differentially regulated proteins including HSP70, ribulose bisphosphate carboxylase/oxygenase activase, plastocyanin and protoporphyrinogen oxidase suggests their potential role in heat tolerance, at flowering growth stage, in field-grown chickpea. This observation supports unaltered physiological and biochemical performance of the heat-tolerant genotypes (Acc#7) relative to the susceptible genotype (Acc#8) in related studies (Makonya et al. 2019). Characterisation of the candidate proteins identified in the current study as well as their specific roles in the tolerance to heat stress in chickpea are integral to further crop improvement initiatives.

Chlorophyll fluorescence and carbohydrate concentration as field selection traits for heat tolerant chickpea genotypes.

Chickpea (Cicer arietinum L.), a cool season crop is severely affected by heat stress, predicted to increase due to warming climates. Research for identifying heat tolerance markers for potential chickpea genotype selection is imperative. The study assessed the response of four chickpea genotypes to a natural temperature gradient in the field using chlorophyll fluorescence, non-structural carbohydrate, chlorophyll concentrations, gas exchange and grain yield. Field experiments were carried out in two winter seasons at three locations with known differences in temperature in NE South Africa. Results showed two genotypes were tolerant to heat stress with an Fv/Fm of 0.83-0.85 at the warmer site, while the two sensitive genotypes showed lower Fv/Fm of 0.78-0.80. Both dark-adapted Fv/Fm and Fq'/Fm' (where Fq' = Fm' -F) measured at comparable high light levels correlated positively with grain yield. The two tolerant genotypes also showed higher photosynthetic rates, starch, sucrose and grain yield than the sensitive genotypes at the warmer site. However, these parameters were consistently higher at the cooler sites than at the warmer. These results were further validated by a climate chamber experiment, where higher Fv/Fm decline in the sensitive compared to tolerant genotypes was observed when they were exposed to short-term heat treatments of 30/25 °C and 35/30 °C. Tolerant genotypes had higher Fv/Fm (0.78-0.81) and grain yield plant-1(1.12-2.37g) compared to sensitive genotypes (0.74-0.75) and (0.32-0.89g plant-1) respectively in the 35/30 °C. It is concluded that chlorophyll fluorescence and leaf carbohydrates are suitable tools for selection of heat tolerant chickpea genotypes under field conditions, while the coolest site showed favourable conditions for chickpea production.

Linking root traits to superior phosphorus uptake and utilisation efficiency in three Fabales in the Core Cape Subregion, South Africa.

In the low-P soil of the fynbos biome, plants have evolved several morphological and physiological P acquisition and use mechanisms, leading to variable uptake and use efficiencies. We expected that plants grown in low-P soils would exhibit greater P acquisition traits and hypothesised that Aspalathus linearis (Burm. f.) R. Dahlgren, a cluster-root-forming species adapted to drier and infertile soils, would be the most efficient at P acquisition compared with other species. Three fynbos Fabales species were studied: A. linearis and Podalyria calyptrata (Retz.) Willd, both legumes, and Polygala myrtifolia L., a nonlegume. A potted experiment was conducted where the species were grown in two soil types with high P (41.18mgkg-1) and low P (9.79mgkg-1). At harvest, biomass accumulation, foliar nutrients and P acquisition mechanisms were assessed. Polygala myrtifolia developed a root system with greater specific root length, root hair width and an average root diameter that exuded a greater amount of citrate and, contrary to the hypothesis, exhibited greater whole-plant P uptake efficiency. However, P. calyptrata had higher P use efficiency, influenced by N availability through N2 fixation. Specific root length, root length and root:shoot ratio were promising morphological traits for efficient foraging of P, whereas acid phosphatase exudation was the best physiological trait for solubilisation of P.

Differential Preference of Burkholderia and Mesorhizobium to pH and Soil Types in the Core Cape Subregion, South Africa.

Over 760 legume species occur in the ecologically-heterogeneous Core Cape Subregion (CCR) of South Africa. This study tested whether the main symbionts of CCR legumes (Burkholderia and Mesorhizobium) are phylogenetically structured by altitude, pH and soil types. Rhizobial strains were isolated from field nodules of diverse CCR legumes and sequenced for 16S ribosomic RNA (rRNA), recombinase A (recA) and N-acyltransferase (nodA). Phylogenetic analyses were performed using Bayesian and maximum likelihood techniques. Phylogenetic signals were determined using the D statistic for soil types and Pagel's λ for altitude and pH. Phylogenetic relationships between symbionts of the narrowly-distributed Indigofera superba and those of some widespread CCR legumes were also determined. Results showed that Burkholderia is restricted to acidic soils, while Mesorhizobium occurs in both acidic and alkaline soils. Both genera showed significant phylogenetic clustering for pH and most soil types, but not for altitude. Therefore, pH and soil types influence the distribution of Burkholderia and Mesorhizobium in the CCR. All strains of Indigofera superba were identified as Burkholderia, and they were nested within various clades containing strains from outside its distribution range. It is, therefore, hypothesized that I. superba does not exhibit rhizobial specificity at the intragenic level. Implications for CCR legume distributions are discussed.

Prioritisation of native legume species for further evaluation as potential forage crops in water-limited agricultural systems in South Africa.

In the face of climate change, identification of forage species suitable for dryland farming under low rainfall conditions in South Africa is needed. Currently, there are only a limited number of forage species suitable for dryland farming under such conditions. The objective of this study was to identify and prioritise native legume species that could potentially be used in dryland farming systems in water-limited agro-ecosystems in South Africa. Using a combination of ecological niche modelling techniques, plant functional traits, and indigenous knowledge, 18 perennial herbaceous or stem-woody legume species were prioritised for further evaluation as potential fodder species within water-limited agricultural areas. These species will be evaluated further for their forage quality and their ability to survive and produce enough biomass under water limitation and poor edaphic conditions.

Biogeographical Patterns of Legume-Nodulating Burkholderia spp.: from African Fynbos to Continental Scales.

UNLABELLED: Rhizobia of the genus Burkholderia have large-scale distribution ranges and are usually associated with South African papilionoid and South American mimosoid legumes, yet little is known about their genetic structuring at either local or global geographic scales. To understand variation at different spatial scales, from individual legumes in the fynbos (South Africa) to a global context, we analyzed chromosomal (16S rRNA, recA) and symbiosis (nifH, nodA, nodC) gene sequences. We showed that the global diversity of nodulation genes is generally grouped according to the South African papilionoid or South American mimosoid subfamilies, whereas chromosomal sequence data were unrelated to biogeography. While nodulation genes are structured on a continental scale, a geographic or host-specific distribution pattern was not detected in the fynbos region. In host range experiments, symbiotic promiscuity of Burkholderia tuberum STM678(T) and B phymatum STM815(T) was discovered in selected fynbos species. Finally, a greenhouse experiment was undertaken to assess the ability of mimosoid (Mimosa pudica) and papilionoid (Dipogon lignosus, Indigofera filifolia, Macroptilium atropurpureum, and Podalyria calyptrata) species to nodulate in South African (fynbos) and Malawian (savanna) soils. While the Burkholderia-philous fynbos legumes (D lignosus, I filifolia, and P calyptrata) nodulated only in their native soils, the invasive neotropical species M pudica did not develop nodules in the African soils. The fynbos soil, notably rich in Burkholderia, seems to retain nodulation genes compatible with the local papilionoid legume flora but is incapable of nodulating mimosoid legumes that have their center of diversity in South America. IMPORTANCE: This study is the most comprehensive phylogenetic assessment of root-nodulating Burkholderia and investigated biogeographic and host-related patterns of the legume-rhizobial symbiosis in the South African fynbos biome, as well as at global scales, including native species from the South American Caatinga and Cerrado biomes. While a global investigation of the rhizobial diversity revealed distinct nodulation and nitrogen fixation genes among South African and South American legumes, regionally distributed species in the Cape region were unrelated to geographic and host factors.

DNA barcodes reveal microevolutionary signals in fire response trait in two legume genera.

Large-scale DNA barcoding provides a new technique for species identification and evaluation of relationships across various levels (populations and species) and may reveal fundamental processes in recently diverged species. Here, we analysed DNA sequence variation in the recently diverged legumes from the Psoraleeae (Fabaceae) occurring in the Cape Floristic Region (CFR) of southern Africa to test the utility of DNA barcodes in species identification and discrimination. We further explored the phylogenetic signal on fire response trait (reseeding and resprouting) at species and generic levels. We showed that Psoraleoid legumes of the CFR exhibit a barcoding gap yielding the combination of matK and rbcLa (matK + rbcLa) data set as a better barcode than single regions. We found a high score (100 %) of correct identification of individuals to their respective genera but a very low score (<50 %) in identifying them to species. We found a considerable match (54 %) between genetic species and morphologically delimited species. We also found that different lineages showed a weak but significant phylogenetic conservatism in their response to fire as reseeders or resprouters, with more clustering of resprouters than would be expected by chance. These novel microevolutionary patterns might be acting continuously over time to produce multi-scale regularities of biodiversity. This study provides the first insight into the DNA barcoding campaign of land plants in species identification and detection of the phylogenetic signal in recently diverged lineages of the CFR.

Psoraleaditurnerae and P.vanberkelae (Psoraleeae, Fabaceae): two new species restricted to the Core Cape Region of South Africa.

Two new species of Psoralea L. are described: Psoraleaditurnerae A. Bello, C.H. Stirt. & Muasya, sp. nov. and Psoraleavanberkelae C.H. Stirt., A. Bello & Muasya, sp. nov. Psoraleaditurnerae is endemic to the Outeniqua mountains (Camferskloof) and is characterised by a mass of numerous basal shoots out of which emerge 2-3 woody stems up to 2 m tall, 3-foliolate needle-like leaflets at the base of the seasonally growing shoot reducing to one towards the apex and bearing numerous 1-3-flowered axillary inflorescences along its length; each mauve to purple and white flower subtended by a trifid cupulum. Psoraleavanberkelae is characterised by its spreading mounding habit, short tightly packed fleshy leaves, with large impressed papillae, densely glandular short broadly triangular stipules, pale to intense mauve to deep blue flowers, standard with a dark purple central blotch above a M-shaped white patch situated above claw, and khaki seeds with purple flecks.

Balanced allocation of organic acids and biomass for phosphorus and nitrogen demand in the fynbos legume Podalyria calyptrata.

Podalyria calyptrata is from fynbos soils with low availability of phosphorus (P) and nitrogen (N). We investigated the physiological basis for tolerance of low P supply in nodulated P. calyptrata and examined responses to increased supply of combined-N as Ca(NO3)2 and P. It was hypothesized that increasing supply of combined-N would stimulate P-acquisition mechanisms and enhance plant growth with high P supply. Biomass, leaf [N] and [P], organic acid and phosphatase root exudates, and phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activity in nodules and roots were examined in two N×P experiments. Low P supply decreased leaf [P] and limited growth, decreasing the nodule:root ratio but increasing nodular PEPC and MDH activity for enhanced P-acquisition or P-utilization. At low P supply, a N-induced demand for P increased root exudation of citrate and PEPC and MDH activity in roots. Greater combined-N supply inhibited nodulation more at low P supply than at high P supply. With a P-induced demand for N the plants nodulated prolifically and increased combined-N supply did not enhance plant growth. The physiological basis for N2-fixing P. calyptrata tolerating growth at low P supply and responding to greater P supply is through balanced acquisition of P and N for plant demand.

Increasing nitrogen supply stimulates phosphorus acquisition mechanisms in the fynbos species Aspalathus linearis.

We investigated the physiological basis for tolerance of limiting P supply and for enhanced growth with simultaneous addition of N and P in Aspalathus linearis (Burm. f.) R. Dahlgren. It was hypothesised that increasing N supply would stimulate P acquisition mechanisms and enhance plant growth with high P supply. In sand, plants received 100µM, 300µM, 500µM and 700µM N at a low P level of 10µM and a high P level of 100µM. In solution, plants received 200µM and 500µM N at a low P level of 5µM and a high P level of 15µM. Cluster roots formed only in plants with low P supply. Roots showed greater citrate and malate production and phosphatase activity at 5µM P than at 15µM P. At 10µM P, greater N supply enhanced cluster root formation to 60% of root biomass, and increased the phosphatase activity of noncluster roots and succinate release by both root types. At a high P supply of 15µM, greater N supply stimulated phosphatase activity of roots by 50%, increasing P uptake and plant growth. With increased resource partitioning towards P acquisition due to greater P demand, A. linearis is tolerant of low P supply and highly responsive to combined addition of N and P.

Effects of UV-B radiation on seed yield of Glycine max and an assessment of F1 generation progeny for carryover effects.

Glycine max (L.) Merr plants were grown outdoors in potted sand exposed to elevated ultraviolet-B (UV-B) radiation provided by filtered fluorescent lamps to determine the effects of UV-B on seed yield and UV-B-induced carryover effects in the F1 generation. Increased UV-B radiation had no detectable effects on reproductive parameters except for a reduction on seed number per plant and an increase in the number of unseeded pods per plant and dry weight of unseeded pods per plant in the field supplemental UV-B experiment. Studies on carryover effects in the greenhouse progeny growth trial also showed no effect of parental treatment with UV-B on biomass production, and most symbiotic-N traits and plant metabolite measured. However, the concentrations of N in nodules and starch in roots were significantly increased in the F1 generation progeny from elevated UV-B radiation relative to their F1 counterparts from ambient radiation. Assessing the effects of seed size on plant growth and symbiotic function in the F1 progeny showed that total biomass, dry matter yield of individual organs (leaves, stems, roots and nodules), total plant N and fixed-N rose with increasing seed size. Seed concentration of flavonoids was also enhanced with increasing seed size. These findings suggest that subtle changes did occur in the F1 generation progeny of parental plants exposed to elevated UV-B with potential to accumulate with further exposure to elevated UV-B radiation.

Responses to ultraviolet-B radiation by purely symbiotic and NO3-fed nodulated tree and shrub legumes indigenous to southern Africa.

Purely symbiotic and NO3-fed nodulated seedlings of Virgilia oroboides (Bergius) T.M. Salter, Cyclopia maculata (L.) Vent and Podalyria calyptrata Willd. were exposed to biologically effective ultraviolet-B radiation (UV-B) to assess the effects of above- and below-ambient UV-B on growth, symbiotic function and metabolite concentrations. Seedlings were grown outdoors either on tables under ambient or 34 or 66% above-ambient UV-B conditions (UV-B100 control, UV-B134 and UV-B166, respectively), or in chambers providing below-ambient (22% of ambient) UV-B (UV-B22) along with a UV-A control and a photosynthetically active radiation (PAR) control. Exposure of seedlings to UV-B166 radiation reduced (P < or = 0.05) leaf and stem dry mass by 34 and 39%, respectively, in C. maculata, and reduced leaf nitrogen concentration (%N) by 12% in V. oroboides. Nodule %N in C. maculata and stem %N in P. calyptrata also decreased (P < or = 0.05) in response to UV-B22 radiation compared with the UV-A control, but not compared with the PAR control. Concentrations of flavonoids, soluble sugars and starch were unaltered by the UV-B treatments. Application of 1 mM NO3 to UV-B166-treated seedlings increased whole-plant dry mass of V. oroboides and P. calyptrata by 47 and 52%, respectively. Dry mass of organs, nodule %N and total N concentration of these species also increased with NO3 application. However, NO3 supply decreased (P < or = 0.05) nodule dry mass, stem %N and leaf %N as well as root and leaf anthocyanin concentrations in C. maculata. In terms of UV-B x N interactions, dry mass of stems, roots, nodules and total biomass of NO3-fed C. maculata seedlings were reduced, and nodule %N, total N and leaf anthocyanins were depressed by the UV-B134 and UV-B166 treatments relative to UV-B100-treated seedlings. Although we found that above-ambient UV-B had no effects on growth and symbiotic function of V. oroboides and P. calyptrata seedlings, feeding NO3 to these species increased (P < or = 0.05) seedling growth. In contrast, purely symbiotic C. maculata seedlings were sensitive to the UV-B166 radiation treatment, and adding NO3 further increased their sensitivity to both the UV-B134 and UV-B166 treatments.

Response of purely symbiotic and NO3-fed nodulated plants of Lupinus luteus and Vicia atropurpurea to ultraviolet-B radiation.

The effects of elevated UV-B radiation on growth, symbiotic function and concentration of metabolites were assessed in purely symbiotic and NO3-fed nodulated plants of Lupinus luteus and Vicia atropurpurea grown outdoors either on tables under supplemental UV-B radiation or in chambers covered with different types of plexi-glass to attenuate solar ultraviolet radiation. Moderately and highly elevated UV-B exposures simulating 15% and 25% ozone depletion as well as sub- ambient UV-B did not alter organ growth, plant total dry matter and N content per plant in both L. luteus and V. atropurpurea. In contrast, elevated UV-B increased (P <0.05) flavonoid and anthocyanin concentrations in roots and leaves of L. luteus, but not of V. atropurpurea. Feeding nodulated plants of L. luteus under elevated UV-B radiation with 2 mM NO3 increased (P <0.05) nodule, leaf and total dry matter, and whole plant N content. With V. atropurpurea, NO3 reduced (P <0.05) nodule activity, root %N and concentrations of flavonoids, anthocyanins in roots and leaves and soluble sugars in roots, in contrast to an observed increase (P <0.05) in nodule dry matter per plant. Similarly, supplying 2 mM NO3 to L. luteus plants exposed to sub-ambient UV-B radiation significantly reduced individual organ growth, plant total biomass, nodule dry matter, nodule %N, and whole plant N content, as well as root concentrations of flavonoids, anthocyanins, soluble sugars, and starch of L. luteus, but not V. atropurpurea plants. These results show no adverse effect of elevated UV-B radiation on growth and symbiotic function of L. luteus and V. atropurpurea plants. However, NO3 supply promoted growth in L. luteus plants exposed to the highly elevated UV-B radiation.

Effects of UV-B radiation on plant growth, symbiotic function and concentration of metabolites in three tropical grain legumes.

Vigna unguiculata (L.) Walp. (cowpea), Glycine max (L.) Merr (soybean) and Phaseolus vulgaris (L.) (common bean) plants were exposed to UV-B radiation at above- and below-ambient levels, and their effects on growth, symbiotic performance and root concentration of metabolites were assessed. Moderately and highly elevated UV-B exposures averaging 32 and 62% above ambient had no effect on plant total dry matter, nodule number, nodule mass, nodule size, N fixed or root concentration of flavonoids, anthocyanins, soluble sugars and starch in the three species studied. However, N concentrations were markedly reduced in roots of G. max and P.vulgaris, and in leaves of P. vulgaris, which contrasted with the significant increase in stems and leaves of V.unguiculata. Below-ambient UV-B exposures averaging 22% of ambient also altered growth and metabolism of these legumes. Total plant dry matter, nodule number, nodule dry mass, N fixed and root starch concentrations in V.unguiculata decreased relative to both visible and UV-A radiation controls, whereas in G. max and P. vulgaris, these parameters were not altered. Root concentrations of flavonoids and anthocyanins in all species tested were also unchanged with below-ambient UV-B exposures. Taken together, growth and symbiotic function of these species remained unaltered with exposure to above-ambient UV-B, but differed in their response to below-ambient UV-B radiation.

Effects of elevated ultraviolet-B radiation on native and cultivated plants of southern Africa.

Seventeen herb, shrub and tree species of commercial and ecological importance in southern Africa were exposed at one location to ultraviolet-B (UV-B, 280-315 nm) radiation approx. 35 % above clear-sky background (control). The aims were to assess how UV-B affects canopy area, dry mass, and some biochemical and morphological properties of leaves, and to investigate whether differences between species are related to growth form of the plants. There was no pattern of response to UV-B related to growth form. Leaves of trees had altered chlorophyll a and b, carotenoid and flavonoid concentrations, but those of shrubs or herbs did not. Non-structural carbohydrates were unaffected. Smaller canopy areas and dry masses were observed under enhanced UV-B, but these were not statistically different among growth forms. There was a general insensitivity of species to elevated UV-B. Only five species had significantly altered leaf biochemical and morphological properties, canopy area and dry mass, the changes differing in magnitude. There was no consistent pattern of change in leaf thickness or biochemical composition with increased UV-B. Correlation analyses did not support the view that growth is less negatively affected in species with thick leaves or in those where leaf thickness increases, or in species with naturally high leaf flavonoid contents or that are able to synthesize additional flavonoids in response to UV-B enhancement. The analyses did not support the hypothesis that growth was inhibited by starch accumulation in leaves under elevated UV-B. However, changes in leaf shape did correlate with canopy area and dry mass, showing the importance of photomorphogenetic changes caused by UV-B which affect species' performance. We conclude that generalizations on plant sensitivity to UV-B based on growth form and functional type could be misleading, and that the great majority of economically important species of the region are likely to be insensitive to future UV-B increases. Notable exceptions include the Colophospermum mopane tree ecotypes chota and leslie and the arable annual Vigna unguiculata, both of which are traditional sources of livelihood to rural African populations and of importance to African industry and agriculture.