Regulation of Leaf Senescence by Cytokinin, Sugars, and Light. Effects on NADH-Dependent Hydroxypyruvate Reductase

The aim of this study was to investigate the interactions between cytokinin, sugar repression, and light in the senescence-related decline in photosynthetic enzymes of leaves. In transgenic tobacco (Nicotiana tabacum) plants that induce the production of cytokinin in senescing tissue, the age-dependent decline in NADH-dependent hydroxypyruvate reductase (HPR), ribulose-1,5-bisphosphate carboxylase/oxygenase, and other enzymes involved in photosynthetic metabolism was delayed but not prevented. Glucose (Glc) and fructose contents increased with leaf age in wild-type tobacco and, to a greater extent, in transgenic tobacco. To study whether sugar accumulation in senescing leaves can counteract the effect of cytokinin on senescence, discs of wild-type leaves were incubated with Glc and cytokinin solutions. The photorespiratory enzyme HPR declined rapidly in the presence of 20 mM Glc, especially at very low photon flux density. Although HPR protein was increased in the presence of cytokinin, cytokinin did not prevent the Glc-dependent decline. Illumination at moderate photon flux density resulted in the rapid synthesis of HPR and partially prevented the negative effect of Glc. Similar results were obtained for the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. It is concluded that sugars, cytokinin, and light interact during senescence by influencing the decline in proteins involved in photosynthetic metabolism

The Use of Maleic Hydrazide for Effective Hybridization of Setaria viridis

An efficient method for crossing green foxtail (Setaria viridis) is currently lacking. S. viridis is considered to be the new model plant for the study of C4 system in monocots and so an effective crossing protocol is urgently needed. S. viridis is a small grass with C4-NADP (ME) type of photosynthesis and has the advantage of having small genome of about 515 Mb, small plant stature, short life cycle, multiple tillers, and profuse seed set, and hence is an ideal model species for research. The objectives of this project were to develop efficient methods of emasculation and pollination, and to speed up generation advancement. We assessed the response of S. viridis flowers to hot water treatment (48°C) and to different concentrations of gibberellic acid, abscisic acid, maleic hydrazide (MH), and kinetin. We found that 500 μM of MH was effective in the emasculation of S. viridis, whilst still retaining the receptivity of the stigma to pollination. We also report effective ways to accelerate the breeding cycle of S. viridis for research through the germination of mature as well as immature seeds in optimized culture media. We believe these findings will be of great interest to researchers using Setaria

Variation between rice accessions in photosynthetic induction in flag leaves and underlying mechanisms

Several breeding initiatives have sought to improve flag leaf performance as its health and physiology are closely correlated to rice yield. Previous studies have described natural variation of photosynthesis for flag leaves; however, none has examined their performance under the non-steady-state conditions that prevail in crop fields. Photosynthetic induction is the transient response of photosynthesis to a change from low to high light. Rice flag leaf photosynthesis was measured in both steady- and non-steady-state conditions to characterize natural variation. Between the lowest and highest performing accession, there was a 152% difference for average CO2 assimilation during induction (Ā300), a 77% difference for average intrinsic water use efficiency during induction (iWUEavg), and a 185% difference for the speed of induction (IT50), indicating plentiful variation. No significant correlation was found between steady- and non-steady-state photosynthetic traits. Additionally, measures of neither steady-state nor non-steady-state photosynthesis of flag leaves correlated with the same measures of leaves in the vegetative growth stage, with the exception of iWUEavg. Photosynthetic induction was measured at six [CO2], to determine biochemical and diffusive limitations to photosynthesis in vivo. Photosynthetic induction in rice flag leaves was limited primarily by biochemistry

Mass screening of rice mutant populations at low CO2 for identification of lowered photorespiration and respiration rates

Introduction: Identifying rice (Oryza sativa) germplasm with improved efficiency of primary metabolism is of utmost importance in order to increase yields. One such approach can be attained through screening genetically diverse populations under altered environmental conditions. Growth or treatment under low carbon dioxide (CO2) concentrations can be used as a means of revealing altered leaf photorespiration, respiration and other metabolic variants. Methods: We developed a pipeline for very high throughput treatment of gamma- and ethyl methanesulfonate- (EMS) induced mutant populations of IR64 rice seedlings at very low CO2 for 7 days. 1050 seedlings per batch at 5th leaf stage were exposed to 60 ppm CO2 for the first day and 30 ppm for the remaining three days. Following this, putative candidates were identified by measuring chlorophyll depletion using SPAD. Screening results showed a distinct difference between the mutants and the WTs. Results and discussion: The mean chlorophyll loss in WTs ranged from 65% to 11% respectively, whereas in the mutant lines chlorophyll loss ranged from 0 to 100%, suggesting considerable phenotypic variation. Rice mutants with a reduced chlorophyll reduction (<10%) were identified as ‘Chlorophyll retention mutants’ (CRMs) under low CO2 stress. In total, 1909 mutant lines (14,000 seedlings) were screened for chlorophyll content under 30 ppm CO2, with 26 lines selected for detailed screening. These 26 putative candidates were self-seeded to produce an M5 generation, used to determine the genetic control of the altered response to low CO2. Gas exchange of light and CO2 response revealed that there were significant variations among photosynthetic properties in two selected rice mutants. The CO2 compensation points in the absence of photorespiration and leaf respiration rates were lower than the WTs and anatomical analyses showed that CRM 29 had improved mesophyll cell area. We propose that this approach is useful for generating new material for breeding rice with improved primary metabolism

HD-ZIP IV gene Roc8 regulates the size of bulliform cells and lignin content in rice

The morphology of bulliform cells located on the upper epidermis of leaves is one of the most important cell structures affecting leaf shape. Although many mechanisms regulating the development of bulliform cells have been reported, the fine regulatory mechanisms governing this process have rarely been described. To identify novel components regulating rice leaf morphology, a mutant showing a constitutively rolling phenotype from the seedling stage to flowering, known as crm1-D, was selected for further analysis. Anatomical analyses in crm1-D were attributable to the size reduction of bulliform cells. The crm1-D was controlled by a single dominant nuclear gene. Map-based cloning revealed that Roc8, an HD zipper class IV family member, was responsible for the crm1-D phenotype. Notably, the 50-bp sequence in the 3′-untranslated region (3′-UTR) of the Roc8 gene represses Roc8 at the translational level. Moreover, the roc8 knockdown lines notably increased the size of bulliform cells. A series of assays revealed that Roc8 negatively regulates the size of bulliform cells. Unexpectedly, Roc8 was also observed to positively mediate lignin biosynthesis without incurring a production penalty. The above results show that Roc8 may have a practical application in cultivating materials with high photosynthetic efficiency and low lignin content

Natural diversity in stomatal features of cultivated and wild Oryza species

Background Stomata in rice control a number of physiological processes by regulating gas and water exchange between the atmosphere and plant tissues. The impact of the structural diversity of these micropores on its conductance level is an important area to explore before introducing stomatal traits into any breeding program in order to increase photosynthesis and crop yield. Therefore, an intensive measurement of structural components of stomatal complex (SC) of twenty three Oryza species spanning the primary, secondary and tertiary gene pools of rice has been conducted. Results Extensive diversity was found in stomatal number and size in different Oryza species and Oryza complexes. Interestingly, the dynamics of stomatal traits in Oryza family varies differently within different Oryza genetic complexes. Example, the Sativa complex exhibits the greatest diversity in stomatal number, while the Officinalis complex is more diverse for its stomatal size. Combining the structural information with the Oryza phylogeny revealed that speciation has tended towards increasing stomatal density rather than stomatal size in rice family. Thus, the most recent species (i.e. the domesticated rice) eventually has developed smaller yet numerous stomata. Along with this, speciation has also resulted in a steady increase in stomatal conductance (anatomical, gmax) in different Oryza species. These two results unambiguously prove that increasing stomatal number (which results in stomatal size reduction) has increased the stomatal conductance in rice. Correlations of structural traits with the anatomical conductance, leaf carbon isotope discrimination (∆13C) and major leaf morphological and anatomical traits provide strong supports to untangle the ever mysterious dependencies of these traits in rice. The result displayed an expected negative correlation in the number and size of stomata; and positive correlations among the stomatal length, width and area with guard cell length, width on both abaxial and adaxial leaf surfaces. In addition, gmax is found to be positively correlated with stomatal number and guard cell length. The ∆13C values of rice species showed a positive correlation with stomatal number, which suggest an increased water loss with increased stomatal number. Interestingly, in contrast, the ∆13C consistently shows a negative relationship with stomatal and guard cell size, which suggests that the water loss is less when the stomata are larger. Therefore, we hypothesize that increasing stomatal size, instead of numbers, is a better approach for breeding programs in order to minimize the water loss through stomata in rice. Conclusion Current paper generates useful data on stomatal profile of wild rice that is hitherto unknown for the rice science community. It has been proved here that the speciation has resulted in an increased stomatal number accompanied by size reduction during Oryza’s evolutionary course; this has resulted in an increased gmax but reduced water use efficiency. Although may not be the sole driver of water use efficiency in rice, our data suggests that stomata are a potential target for modifying the currently low water use efficiency in domesticated rice. It is proposed that Oryza barthii can be used in traditional breeding programs in enhancing the stomatal size of elite rice cultivars

Closer vein spacing by ectopic expression of nucleotide-binding and leucine-rich repeat proteins in rice leaves

Key message Elevated expression of nucleotide-binding and leucine-rich repeat proteins led to closer vein spacing and higher vein density in rice leaves. Abstract To feed the growing global population and mitigate the negative effects of climate change, there is a need to improve the photosynthetic capacity and efficiency of major crops such as rice to enhance grain yield potential. Alterations in internal leaf morphology and cellular architecture are needed to underpin some of these improvements. One of the targets is to generate a “Kranz-like” anatomy in leaves that includes decreased interveinal spacing close to that in C4 plant species. As C4 photosynthesis has evolved from C3 photosynthesis independently in multiple lineages, the genes required to facilitate C4 may already be present in the rice genome. The Taiwan Rice Insertional Mutants (TRIM) population offers the advantage of gain-of-function phenotype trapping, which accelerates the identification of rice gene function. In the present study, we screened the TRIM population to determine the extent to which genetic plasticity can alter vein density (VD) in rice. Close vein spacing mutant 1 (CVS1), identified from a VD screening of approximately 17,000 TRIM lines, conferred heritable high leaf VD. Increased vein number in CVS1 was confirmed to be associated with activated expression of two nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. Overexpression of the two NB-LRR genes individually in rice recapitulates the high VD phenotype, due mainly to reduced interveinal mesophyll cell (M cell) number, length, bulliform cell size and thus interveinal distance. Our studies demonstrate that the trait of high VD in rice can be achieved by elevated expression of NB-LRR proteins limited to no yield penalty

A low CO2-responsive mutant of Setaria viridis reveals that reduced carbonic anhydrase limits C4 photosynthesis

In C4 species, β-carbonic anhydrase (CA), localized to the cytosol of the mesophyll cells, accelerates the interconversion of CO2 to HCO3–, the substrate used by phosphoenolpyruvate carboxylase (PEPC) in the first step of C4 photosynthesis. Here we describe the identification and characterization of low CO2-responsive mutant 1 (lcr1) isolated from an N-nitroso-N-methylurea- (NMU) treated Setaria viridis mutant population. Forward genetic investigation revealed that the mutated gene Sevir.5G247800 of lcr1 possessed a single nucleotide transition from cytosine to thymine in a β-CA gene causing an amino acid change from leucine to phenylalanine. This resulted in severe reduction in growth and photosynthesis in the mutant. Both the CO2 compensation point and carbon isotope discrimination values of the mutant were significantly increased. Growth of the mutants was stunted when grown under ambient pCO2 but recovered at elevated pCO2. Further bioinformatics analyses revealed that the mutation has led to functional changes in one of the conserved residues of the protein, situated near the catalytic site. CA transcript accumulation in the mutant was 80% lower, CA protein accumulation 30% lower, and CA activity ~98% lower compared with the wild type. Changes in the abundance of other primary C4 pathway enzymes were observed; accumulation of PEPC protein was significantly increased and accumulation of malate dehydrogenase and malic enzyme decreased. The reduction of CA protein activity and abundance in lcr1 restricts the supply of bicarbonate to PEPC, limiting C4 photosynthesis and growth. This study establishes Sevir.5G247800 as the major CA allele in Setaria for C4 photosynthesis and provides important insights into the function of CA in C4 photosynthesis that would be required to generate a rice plant with a functional C4 biochemical pathway

Key changes in gene expression identified for different stages of C4 evolution in Alloteropsis semialata.

C4 photosynthesis is a complex trait that boosts productivity in tropical conditions. Compared to C3 species, the C4 state seems to require numerous novelties, but species comparisons can be confounded by long divergence times. Here, we exploit the photosynthetic diversity that exists within a single species, the grass Alloteropsis semialata, to detect changes in gene expression associated with different photosynthetic phenotypes. Phylogenetically-informed comparative transcriptomics show that intermediates with a weak C4 cycle are separated from the C3 phenotype by increases in the expression of 58 genes (0.22% of genes expressed in the leaves), including those encoding just three core C4 enzymes: ASP-AT, PCK, and PEPC. The subsequent transition to full C4 physiology was accompanied by increases in another 15 genes (0.06%), including only the core C4 enzyme PPDK. These changes likely created a rudimentary C4 physiology, and isolated populations subsequently improved this emerging C4 physiology, resulting in a patchwork of expression for some C4-accessory genes. Our work shows how C4 assembly in A. semialata happened in incremental steps, each requiring few alterations over the previous one. These create short bridges across adaptive landscapes that likely facilitated the recurrent origins of C4 photosynthesis through a gradual process of evolution