Patrick J. Rich

Chemically Induced Cuticle Mutation Affecting Epidermal Conductance to Water Vapor and Disease Susceptibility in Sorghum bicolor (L.) Moench

Analysis of Sorghum bicolor bloomless (bm) mutants with altered epicuticular wax (EW) structure uncovered a mutation affecting both EW and cuticle deposition. The cuticle of mutant bm-22 was about 60% thinner and approximately one-fifth the weight of the wild-type parent P954035 (WT-P954035) cuticles. Reduced cuticle deposition was associated with increased epidermal conductance to water vapor. The reduction in EW and cuticle deposition increased susceptibility to the fungal pathogen Exserohilum turcicum. Evidence suggests that this recessive mutation occurs at a single locus with pleiotropic effects. The independently occurring gene mutations of bm-2, bm-6, bm-22, and bm-33 are allelic. These chemically induced mutants had essentially identical EW structure, water loss, and cuticle deposition. Furthermore, 138 F2 plants from a bm-22 x WT-P954035 backcross showed no recombination of these traits. This unique mutation in a near-isogenic background provides a useful biological system to examine plant cuticle biosynthesis, physiology, and function.

Salinity, Osmolytes and Compatible Solutes

In order to maintain low cytosolic Na+, and cytosolic K+ concentrations within narrow limits (100–150 mM) across a broad range of external and vacuolar concentrations of NaCl, it is essential that other solutes be accumulated in the cytoplasm to keep this compartment in osmotic balance with the external medium and vacuole. Such solutes should be osmolytes that are non-toxic and “compatible” with cytoplasmic enzymes over wide concentration ranges. A number of such solutes have been identified, their pathways of synthesis elucidated, and progress has been in isolating genes encoding key enzymes of their biosynthetic pathways.

Near-Isogenic Lines of Maize Differing for Glycinebetaine

A series of near-isogenic glycinebetaine-containing and -deficient F8 pairs of Zea mays L. (maize) lines were developed. The pairs of lines differ for alternative alleles of a single locus; the wild-type allele conferring glycinebetaine accumulation is designated Bet1 and the mutant (recessive) allele is designated bet1. The near-isogenic lines were used to investigate whether glycinebetaine deficiency affects the pool size of the glycinebetaine precursor, choline, using a new method for glycinebetaine and choline determination: stable isotope dilution plasma desorption mass spectrometry. Glycinebetaine deficiency in maize was associated with a significant expansion of the free choline pool, but the difference in choline pool size was not equal to the difference in glycinebetaine pool size, suggesting that choline must down-regulate its own synthesis. Consistent with this, glycinebetaine deficiency was also associated with the accumulation of the choline precursor, serine. A randomly amplified polymorphic DNA marker was identified that detects the bet1 allele. In 62 F8 families tested the 10-mer primer 5[prime]-GTCCTCGTAG produced a 1.2-kb polymerase chain reaction product only when DNA from Bet1/bet1 or bet1/bet1 lines was used as template. All 26 homozygous Bet1/Bet1 F8 families tested were null for this marker.

Resistance to Striga hermonthica in a maize inbred line derived from Zea diploperennis.

Breeding for resistance to Striga in maize (Zea mays), with paucity of donor source and known mechanisms of resistance, has been challenging. Here, post-attachment development of S. hermonthica was monitored on two maize inbreds selected for field resistance and susceptibility reactions to Striga at the International Institute of Tropical Agriculture. Haustorial invasion of the parasite into roots of these inbreds was examined histologically. Morphological differences were observed between roots of the susceptible and the resistant inbreds. The resistant maize had fewer Striga attachments, delayed parasitic development and higher mortality of attached parasites compared with the susceptible inbred. Striga on the susceptible inbred usually penetrated the xylem and showed substantial internal haustorial development. Haustorial ingress on the resistant inbred was often stopped at the endodermis. Parasites able to reach resistant host xylem vessels showed diminished haustorial development relative to those invading susceptible roots. These results suggest that the resistant inbred expresses a developmental barrier and incompatible response against Striga parasitism.

Epicuticular Wax Morphology of Bloomless (bm) Mutants in Sorghum bicolor

Sorghum bicolor mutants for cuticular wax production provide a model system for analysis of epicuticular wax (EW) physiology, biochemistry, and genetics. Mutants produced from seeds treated with the chemical mutagens diethyl sulfate (DES) and ethyl methanesulfonate (EMS) were selected in the M2 generation and self-pollinated to produce near-isogenic mutants of two classes: bloomless (lacking visible EW) and sparse bloom (possessing little visible EW). Scanning electron microscopy was used to further divide 33 selected lines into 14 unique classes based on altered EW structure. Mutations have affected the structure of cork silica (CS) cell associated EW or both CS cell and cuticle EW. The resulting spectrum of altered EW structure indicates unique alterations in EW biosynthesis or deposition which may correlate with specific EW alleles and loci within the Sorghum genome.

Towards effective resistance to Striga in African maize

The fascinating biology of Striga parasitism is manifest through a series of signal exchanges between the parasite and its host. As an obligate root hemi-parasite, Striga development is cued to exudates and solutes of host roots but with negative ramifications on host plant health. Striga control in crops, via a variety of biotechnological approaches, needs to be based on increased understanding of this intricate biology. Maize has become the major cereal crop of Africa. However, this New World transplant has shown a paucity of Striga resistance characters relative to native sorghum. In this paper, we review growing evidence for maize genetic defenses against early pre-emergent phases of the Striga life cycle, when the tolls of parasitism are first manifest. Resistance characters first described in maize wild relatives have now been captured in Zea mays. The possible stacking of new and complementary sources of resistance in improved maize varieties targeted for Striga prone areas is discussed. An integrated approach combining genetic with other control measures is advocated with a more realistic view of the resource challenges prevalent in African agriculture.

Involvement of Cork Cells in the Secretion of Epicuticular Wax Filaments on Sorghum bicolor (L.) Moench

Tubular epicuticular wax (EW) filaments on Sorghum bicolor were shown to be secreted from smooth conical papillae within the apical walls of epidermal cork cells. Ultrastructural changes during light-induced EW secretion were examined in wild-type plants and near-isogenic mutants with reduced total EW deposition. Our results indicated that cork cell ER membranes were involved in the production of epicuticular wax precursors (EWPs). The density of ER increased during light exposure and preceded EW synthesis. The increase in ER was directly related to total EW deposition on wild-type and mutant abaxial sheaths. The orientation of ER membranes toward papillae secretion sites indicated that EWP may undergo ER-mediated directional transport. The high vesicle density in cytoplasmic extensions under papillae indicated that EWPs were vesiculated for exocytosis at the papillar secretion sites. Osmiophilic globules did not appear to be direct EWPs as previously reported. Osmiophilic globules in cork cells were never present in cell walls, cuticles, vesicles, or preferentially associated with ER; globules were randomly dispersed in the cytoplasm and rarely present during the EW-induction period. Distinct microchannels or pores were not evident in the cell wall or cuticle layers, indicating that EWPs diffused to the surface. Wall swellings near the base of papillae where a dense-staining wall modification first contacts the cuticle and where EW filaments emerge indicate a potential preferred pathway for EWP transport. An osmiophilic layer within apical cork cell walls appears to function in EW secretion; however, its exact role is yet unclear.

Leaf sheath cuticular waxes on bloomless and sparse-bloom mutants of Sorghum bicolor

Leaf sheath cuticular waxes on wild-type Sorghum bicolor were approximately 96% free fatty acids, with the C28 and C30 acids being 77 and 20% of these acids, respectively. Twelve mutants with markedly reduced wax load were characterized for chemical composition. In all of the 12 mutants, reduction in the amount of C28 and C30 acids accounted for essentially all of the reduction in total wax load relative to wildtype. The bm2 mutation caused a 99% reduction in total waxes. The bm4, bm5, bm6, bm7 and h10 mutations caused more than 91% reduction in total waxes, whereas the remaining six mutants, bm9, bm11, h7, h11, h12 and h13, caused between 35 and 78% reduction in total wax load. Relative to wild-type, bm4 caused a large increase in the absolute amount of C22, C24 and C26 acids, and reduction in the C28 and longer acids, suggesting that bm4 may suppress elongation of C26, acyl-CoA primarily. The h10 mutation increased the absolute amounts of the longest chain length acids, but reduced shorter acids, suggesting that h10 may suppress termination of acyl-CoA elongation. The bm6, bm9, bm11, h7, h11, h12 and h13 mutations increased the relative amounts, but not absolute amounts, of longer chain acids. Based on chemical composition alone, it is still uncertain which genes and their products were altered by these mutations. Nevertheless, these Sorghum cuticular wax mutants should provide a valuable resource for future studies to elucidate gene involvement in the biosynthesis of cuticular waxes, in particular, the very-long-chain fatty acids.

Scattering of ultraviolet and photosynthetically active radiation by sorghum bicolor: influence of epicuticular wax

Near-isogenic mutants of Sorghum bicolor with genetic alterations affecting epicuticular wax (EW) structure but having similar canopy architecture provided a model system to examine the influence of EW on plant radiation scattering. Differences in canopies with two different sheath EW amounts showed differences in angular reflectance and transmittance. The differences varied with waveband of radiation. Canopy ultraviolet-B (UVB) and photosynthetically active radiation (PAR) backward reflectance in the principal solar plane were higher by wild-type plants (N-15) bearing reflective stalk EW filaments than mutant plants (bm-15) lacking stalk EW filaments. Between panicle emergence to anthesis the backward PAR reflectance increased more in the N-15 than bm-15 canopy. We suspect that the increase was a result of reflections from stalk facets emerging above the surface plane of the canopy foliage and exposing reflective EW. As panicles emerged above the foliage, canopy UVB and PAR forward reflectance by bm-15 increased while forward reflectance by N-15 decreased. The increased forward reflectance from bm-15 may be because of high specular reflectance from the microscopically smooth bm-15 stalk surfaces. Based on comparisons of probability distributions, significant differences in PAR and UVB canopy transmittance were detected between N-15 and bm-15. The median UVB transmittance was greater in the bm-15 canopy than the N-15 canopy, while the median PAR transmittance was the same for the two canopies. The greater transmittance in the N-15 canopy corresponded with lower EW load of the sheaths, but the difference between canopies was within the experimental error. Distinct influences of the stalk EW on canopy reflectance and transmittance were difficult to assess because of the relatively low proportion of surface area containing EW, the experimental errors associated with UVB irradiance field measurements. The optical properties of the S. bicolor canopy varied by waveband. Results suggested that the canopies both had a higher UVB transmittance than PAR in the shaded fraction, due in part to the greater proportion of sky radiation in the global UVB over the PAR waveband and possibly in part as a result of differences in scattering by the canopy. Distinct differences were difficult to assess because of the experimental error and differences in diffuse fraction of UVB and PAR under clear sky conditions. The empirical probability distributions of canopy transmittance in both UVB and PAR were non-gaussian; fitting the β distribution better than the gaussian distribution. It is suggested that the irradiance in sunlit and shaded fractions not be described using statistics from the normal distribution.

Mutation in sorghum LOW GERMINATION STIMULANT 1 alters strigolactones and causes Striga resistance

Significance The parasitic weed Striga is the greatest biological constraint to production of many crops in Africa and parts of Asia. Genetic control is the most feasible means of crop protection from this pest. We report on identification of a gene regulating Striga resistance in sorghum and the associated change in strigolactone chemistry. Knowing this gene and its various natural alleles, sorghum breeders can design markers within it to facilitate its transfer into improved varieties providing farmers effective control of Striga in infested fields. The gene could also be used to potentially improve Striga resistance through genome editing in crops such as maize that evolved away from Striga, and hence have a paucity of Striga resistance genes. Striga is a major biotic constraint to sorghum production in semiarid tropical Africa and Asia. Genetic resistance to this parasitic weed is the most economically feasible control measure. Mutant alleles at the LGS1 (LOW GERMINATION STIMULANT 1) locus drastically reduce Striga germination stimulant activity. We provide evidence that the responsible gene at LGS1 codes for an enzyme annotated as a sulfotransferase and show that functional loss of this gene results in a change of the dominant strigolactone (SL) in root exudates from 5-deoxystrigol, a highly active Striga germination stimulant, to orobanchol, an SL with opposite stereochemistry. Orobanchol, although not previously reported in sorghum, functions in the multiple SL roles required for normal growth and environmental responsiveness but does not stimulate germination of Striga. This work describes the identification of a gene regulating Striga resistance and the underlying protective chemistry resulting from mutation.