Soybean Leaf Senescence Research Articles

The mechanism of low blue light-induced leaf senescence mediated by GmCRY1s in soybean

Leaf senescence is a crucial trait that has a significant impact on crop quality and yield. Previous studies have demonstrated that light is a key factor in modulating the senescence process. However, the precise mechanism by which plants sense light and control senescence remains largely unknown, particularly in crop species. In this study, we reveal that the reduction in blue light under shading conditions can efficiently induce leaf senescence in soybean. The blue light receptors GmCRY1s rather than GmCRY2s, primarily regulate leaf senescence in response to blue light signals. Our results show that GmCRY1s interact with DELLA proteins under light-activated conditions, stabilizing them and consequently suppressing the transcription of GmWRKY100 to delay senescence. Conversely, LBL reduces the interaction between GmCRY1s and the DELLA proteins, leading to their degradation and premature senescence of leaves. Our findings suggest a GmCRY1s-GmDELLAs-GmWRKY100 regulatory cascade that is involved in mediating LBL-induced leaf senescence in soybean, providing insight into the mechanism of how light signals regulate leaf senescence. Additionally, we generate GmWRKY100 knockout soybeans that show delayed leaf senescence and improved yield under natural field conditions, indicating potential applications in enhancing soybean production by manipulating the leaf senescence trait.

Integrative Inference of Atore1 Mutant Networks in Hormonal Balance Yields Induces Distinct Novel H2o2-Nacs Signaling in Regulating the Onset of Leaf Senescence in Soybean (Glycine Max L.)

Integrative Inference of Atore1 Mutant Networks in Hormonal Balance Yields Induces Distinct Novel H2o2-Nacs Signaling in Regulating the Onset of Leaf Senescence in Soybean (Glycine Max L.)

A Potential Role of Coumestrol in Soybean Leaf Senescence and Its Interaction With Phytohormones.

Coumestrol is a natural organic compound synthesized in soy leaves and functions as a phytoalexin. The coumestrol levels in plants are reported to increase upon insect attack. This study investigates the correlation between coumestrol, senescence, and the effect of phytohormones on the coumestrol levels in soybean leaves. Our analysis involving high-performance liquid chromatography and 2-D gel electrophoresis indicated a significant difference in the biochemical composition of soybean leaves at various young and mature growth stages. Eight chemical compounds were specifically detected in young leaves (V1) only, whereas three different coumestans isotrifoliol, coumestrol, and phaseol were detected only in mature, yellow leaves of the R6 and R7 growth stage. MALDI-TOF-MS analysis was used to identify two proteins 3,9 -dihydroxypterocarpan 6A-monooxygenase (CYP93A1) and isoflavone reductase homolog 2 (IFR2) only in mature leaves, which are key components of the coumestrol biosynthetic pathway. This indicates that senescence in soybean is linked to the accumulation of coumestrol. Following the external application of coumestrol, the detached V1-stage young leaves turned yellow and showed an interesting development of roots at the base of the midrib. Additionally, the application of phytohormones, including SA, methyl jasmonate (MeJA), and ethephon alone and in various combinations induced yellowing within 5 days of the application with a concomitant significant increase in endogenous coumestrol accumulation. This was also accompanied by a significant increase in the expression of genes CYP81E28 (Gm08G089500), CYP81E22 (Gm16G149300), GmIFS1, and GmIFS2. These results indicate that various coumestans, especially coumestrol, accumulate during leaf maturity, or senescence in soybean.

Further insights into how low-light signaling delays leaf senescence in soybean under high-temperature

Plants go through several physiological modifications in their life cycle. Leaf senescence (LS) occurs at the later stage of plant development but can also be initiated by high temperature (HT) stress. LS involves the breakdown of certain key developmental processes in plants, which affect leaf greenness, thereby decreasing plant photosynthetic performance. Light signaling pathway modulates plant growth processes under high-temperature stress, suggesting that light and temperature signaling pathways are linked. However, our knowledge of how light signaling pathways delay LS in soybean under HT remains incomplete. Therefore, this study aimed to investigate how low light signaling pathway delays LS in soybean under HT stress. The results showed that low light signaling delayed LS in soybean under high-temperature stress. Our gas exchange analysis showed that low light signaling improved soybean photosynthetic process and chlorophyll contents under HT stress. Further analysis indicated that low light treatment improved soybean soluble sugar, starch, and free amino acids of plants exposed to HT stress. The phytohormones analysis suggested that under low light, plants accumulated endogenous jasmonic acid (JA) and salicylic acid (SA) when exposed to HT stress. The transcriptional analysis suggested that soybean STAY-GREEN genes (GmSGR1and GmSGR2), sucrose phosphate synthase genes (GmSPS1 and GmSPS2), and starch synthase genes (GmAGP1 and GmUGP1−1) were upregulated in the high-temperature and low light treatment. Again, the JA defense pathway genes GmPDF1.2, GmJAR1.1 GmFAD3.1, and GmPR4.2 expression levels were significantly upregulated in the high-temperature and low light treatment as compared to SA defense pathway genes GmSID2, GmEDS5, GmNPR1, and GmPR1. The regulation mechanisms involving how low light signaling delays leaf senescence in soybean under high temperature through improved photosynthetic rate and JA defense signaling activation are discussed.

Quantification of soybean leaf senescence and maturation as impacted by soil‐ and foliar‐applied nitrogen

The objectives of this study were to: (i) evaluate the effect of various rates of soil‐ and foliar‐applied N at different growth stages on the maturation of soybeans; and (ii) quantify the effect on soybean yield and quality (protein and oil) of soil and foliar N application. Earn 1 CEU in Nutrient Management by reading this article and taking the quiz at www.certifiedcropadviser.org/education/classroom/classes/693.

Quantification of Soybean Leaf Senescence and Maturation as Impacted by Soil- and Foliar-Applied Nitrogen

Quantification of Soybean Leaf Senescence and Maturation as Impacted by Soil- and Foliar-Applied Nitrogen

Coupling of gel-based 2-DE and 1-DE shotgun proteomics approaches to dig deep into the leaf senescence proteome of Glycine max

Leaf senescence is the last stage of leaf development that re-mobilizes nutrients from the source to sink. Here, we have utilized the soybean as a model system to unravel senescence-associated proteins (SAPs). A comparative proteomics approach was used at two contrasting stages of leaf development, namely mature (R3) and senescent (R7). Selection criteria for these two stages were the contrasting differences in their biochemical parameters – chlorophyll, carotenoids and malondialdehyde contents. Proteome analysis involved subjecting the total leaf proteins to 15% poly-ethylene glycol (PEG) pre-fractional method to enrich the low-abundance proteins (LAPs) and their analyses by gel-based 2-DE and 1-DE shotgun proteomics approaches. 2-DE profiling of PEG-supernatant and -pellet fractions detected 153 differential spots between R3 and R7 stages, of which 102 proteins were identified. In parallel, 1-DE shotgun proteomics approach identified 598 and 534 proteins in supernatant and pellet fractions of R3 and R7 stages, respectively. MapMan and Gene Ontology analyses showed increased abundance and/or specific accumulation of proteins related to jasmonic acid biosynthesis and defense, while proteins associated with photosynthesis and ROS-detoxification were decreased during leaf senescence. These findings and the generated datasets further our understanding on leaf senescence at protein level, providing a resource for the scientific community. Biological significanceLeaf senescence is a major biological event in the life cycle of plants that leads to the recycling of nutrients. However, the molecular mechanisms underlying leaf senescence still remain poorly understood. Here, we used a combination of gel-based 2-DE and 1-DE shotgun proteomics approaches to dig deeper into the leaf senescence proteome using soybean leaf as a model experimental material. For the identification of low-abundance proteins, polyethylene glycol (PEG) fractionation was employed and both PEG-supernatant and -pellet fractions were utilized for 2-DE and shotgun proteomic analysis. A total of 1234 (102 from 2-DE and 1132 from 1-DE shotgun proteome analysis) proteins were identified which were functionally annotated using GO and MapMan bioinformatics tools. Our results also emphasize the role of jasmonic acid in soybean leaf senescence.

Blue Light-Dependent Interaction between Cryptochrome2 and CIB1 Regulates Transcription and Leaf Senescence in Soybean

Cryptochromes are blue light receptors that regulate light responses in plants, including various crops. The molecular mechanism of plant cryptochromes has been extensively investigated in Arabidopsis thaliana, but it has not been reported in any crop species. Here, we report a study of the mechanism of soybean (Glycine max) cryptochrome2 (CRY2a). We found that CRY2a regulates leaf senescence, which is a life history trait regulated by light and photoperiods via previously unknown mechanisms. We show that CRY2a undergoes blue light-dependent interaction with the soybean basic helix-loop-helix transcription activator CIB1 (for cryptochrome-interacting bHLH1) that specifically interacts with the E-box (CANNTG) DNA sequences. Analyses of transgenic soybean plants expressing an elevated or reduced level of the CRY2a or CIB1 demonstrate that CIB1 promotes leaf senescence, whereas CRY2a suppresses leaf senescence. Results of the gene expression and molecular interaction analyses support the hypothesis that CIB1 activates transcription of senescence-associated genes, such as WRKY DNA BINDING PROTEIN53b (WRKY53b), and leaf senescence. CIB1 interacts with the E-box-containing promoter sequences of the WRKY53b chromatin, whereas photoexcited CRY2a interacts with CIB1 to inhibit its DNA binding activity. These findings argue that CIB-dependent transcriptional regulation is an evolutionarily conserved CRY-signaling mechanism in plants, and this mechanism is opted in evolution to mediate light regulation of different aspects of plant development in different plant species.

Effects of fully open-air [CO<sub>2</sub>] elevation on leaf ultrastructure, photosynthesis, and yield of two soybean cultivars

The objective of this study was to investigate the effect of elevated (550 ± 17 μmol mol−1) CO2 concentration ([CO2]) on leaf ultrastructure, leaf photosynthesis and seed yield of two soybean cultivars [Glycine max (L.) Merr. cv. Zhonghuang 13 and cv. Zhonghuang 35] at the Free-Air Carbon dioxide Enrichment (FACE) experimental facility in North China. Photosynthetic acclimation occurred in soybean plants exposed to long-term elevated [CO2] and varied with cultivars and developmental stages. Photosynthetic acclimation occurred at the beginning bloom (R1) stage for both cultivars, but at the beginning seed (R5) stage only for Zhonghuang 13. No photosynthetic acclimation occurred at the beginning pod (R3) stage for either cultivar. Elevated [CO2] increased the number and size of starch grains in chloroplasts of the two cultivars. Soybean leaf senescence was accelerated under elevated [CO2], determined by unclear chloroplast membrane and blurred grana layer at the beginning bloom (R1) stage. The different photosynthesis response to elevated [CO2] between cultivars at the beginning seed (R5) contributed to the yield difference under elevated [CO2]. Elevated [CO2] significantly increased the yield of Zhonghuang 35 by 26% with the increased pod number of 31%, but not for Zhonghuang 13 without changes of pod number. We conclude that the occurrence of photosynthetic acclimation at the beginning seed (R5) stage for Zhonghuang 13 restricted the development of extra C sink under elevated [CO2], thereby limiting the response to elevated [CO2] for the seed yield of this cultivar.

Ethylene production under high temperature stress causes premature leaf senescence in soybean

Leaf senescence in soybean (Glycine max L. Merr.) occurs during the later stages of reproductive development and can be triggered or enhanced by high temperature (HT) stress. Ethylene production can trigger premature leaf senescence, but it is unclear whether HT stress produces ethylene and the subsequent influence on physiology and yield of soybean is also uncertain. We hypothesised that ethylene produced under HT stress is involved in premature leaf senescence and that use of an ethylene perception inhibitor would influence physiology and yield. Objectives of this study were to (1) quantify HT-stress-induced ethylene production; (2) quantify effects of HT stress and application of an ethylene perception inhibitor (1-methylcyclopropene; 1-MCP) on source strength traits such as photosynthetic rate, oxidant production, membrane damage and sugar accumulation; and (3) evaluate efficacy of 1-MCP on minimising HT-stress-induced effects on physiological and yield traits. Soybean plants were exposed to HT (38/28°C) or optimum temperature (OT, 28/18°C) for 14 days at the beginning of pod set. Plants at each temperature were treated with 1 μg L–1 1-MCP or left untreated (control). HT stress enhanced ethylene production rates in leaves and pods by 3.2- and 2.1-fold over OT. HT stress decreased photochemical efficiency (5.8%), photosynthetic rate (12.7%), sucrose content (21.5%), superoxide dismutase (13.3%), catalase (44.6%) and peroxidase (42.9%) enzymes activity and increased superoxide radical (63%) and hydrogen peroxide (70.4%) content and membrane damage (54.7%) compared with OT. Application of 1-MCP decreased ethylene production rate and premature leaf senescence traits by enhancing the antioxidant defence system. HT stress decreased seed set percentage (18.6%), seed size (64.5%) and seed yield plant–1 (71.4%) compared with OT, however, foliar spray of 1-MCP increased the seed set percent and seed size, which resulted in a higher yield than the unsprayed control. The present study showed HT stress increased ethylene production rate, which triggered premature leaf senescence, whereas 1-MCP application reduced or postponed premature leaf senescence traits by inhibiting ethylene production.

The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco

The ER-resident molecular chaperone BiP (binding protein) was overexpressed in soybean. When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential. During exposure to drought the stomata of the transgenic lines did not close as much as in the wild type, and the rates of photosynthesis and transpiration became less inhibited than in the wild type. These parameters of drought resistance in the BiP overexpressing lines were not associated with a higher level of the osmolytes proline, sucrose, and glucose. It was also not associated with the typical drought-induced increase in root dry weight. Rather, at the end of the drought period, the BiP overexpressing lines had a lower level of the osmolytes and root weight than the wild type. The mRNA abundance of several typical drought-induced genes [NAC2, a seed maturation protein (SMP), a glutathione-S-transferase (GST), antiquitin, and protein disulphide isomerase 3 (PDI-3)] increased in the drought-stressed wild-type plants. Compared with the wild type, the increase in mRNA abundance of these genes was less (in some genes much less) in the BiP overexpressing lines that were exposed to drought. The effect of drought on leaf senescence was investigated in soybean and tobacco. It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought. BiP overexpressing tobacco and soybean showed delayed leaf senescence during drought. BiP antisense tobacco plants, conversely, showed advanced leaf senescence. It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning. The delay in leaf senescence by BiP overexpression might relate to the absence of the response to drought.

Cloning and Preliminary Characterization of Three Receptor-like Kinase Genes in Soybean

Leaf senescence that occurs in the last stage of leaf development is a genetically programmed process. It is very significant to isolate the upstream components in the senescence signaling pathway and to elucidate the molecular mechanisms that control the initiation and progression of leaf senescence. In this study, full-length cDNAs of three receptor-like protein kinase genes, designated rlpk1, rlpk2 and rlpk3, were cloned from artificially-induced senescent soybean (Glycine max L.) primary leaves (GenBank accession AY687390, AY687391, AF338813). The deduced amino acid sequences indicated that they belonged to a receptor-like kinase family. Each of rlpk1 and rlpk2 encodes a leucine-rich repeat (LRR) receptor-like protein kinase. They both comprise a typical signal peptide, several LRR motifs, a single-pass transmem-brane domain, and a cytoplasmic protein kinase domain. No typical extracellular domain of RLPK3 was predicted. Organ-specific expression pattern analysis by reverse-transcription polymerase chain reaction (RT-PCR) revealed higher expression levels of the three genes in cotyledons, roots and flowers. Phylogenetic analysis indicated that RLPK1 and RLPK2 belonged to an independent branch, whereas RLPK3 shared common nodes with several known RLKs responding to abiotic and biotic stresses. The evident alternations of expression profiles of rlpk1 and rlpk2 induced by the artificial senescence-inducing treatment implied involvements of these two RLKs in regulating soybean leaf senescence. (Managing editor: Li-Hui Zhao)

Identification and functional characterization of a leucine-rich repeat receptor-like kinase gene that is involved in regulation of soybean leaf senescence

We report here the cloning and characterization of a soybean receptor-like kinase (RLK) gene, designated GmSARK (Glycine max senescence-associated receptor-like kinase), which is involved in regulating leaf senescence. The conceptual protein product of GmSARK contains typical domains of LRR receptor-like kinases: a cytoplasmic domain with all the 11 kinase subdomains, a transmembrane domain and an extracelullar domain containing 9 Leucine-Rich Repeat (LRR) units that may act as a receptor. The expression of GmSARK in soybean leaves was up-regulated in all the three tested senescence systems: senescing cotyledons, dark-induced primary leaf senescence and the natural leaf senescence process after florescence. Furthermore, the RNA interference (RNAi)-mediated knocking-down of GmSARK dramatically retarded soybean leaf senescence. A more complex thylakoid membrane system, higher foliar level of chlorophyll content and a very remarkable delay of senescence-induced disintegration of chloroplast structure were observed in GmSARK-RNAi transgenic leaves. A homolog of maize lethal leaf-spot 1 gene, which has been suggested to encode a key enzyme catalyzing chlorophyll breakdown, was isolated and nominated Gmlls1. The expression level of Gmgtr1 gene, which encodes a key enzyme of chlorophyll synthesis, was also analyzed. It was found that Gmlls1 was up-regulated and Gmgtr1 was down-regulated during senescence in wild-type soybean leaves. However, both of the up-regulation of Gmlls1 and down-regulation of Gmgtr1 were retarded during senescence of GmSARK-RNAi transgenic leaves. In addition, over-expression of the GmSARK gene greatly accelerated the senescence progression of CaMV 35S:GmSARK transgenic plants. Taken together, these results strongly suggested the involvement of this LRR-RLK in regulation of soybean leaf senescence, maybe via regulating chloroplast development and chlorophyll accumulation. Multiple functions of GmSARK besides its regulation of leaf senescence were also discussed.

Cloning and expressional characterization of soybean GmLls1 gene

Maize Lls1 (lethal leaf-spot 1) gene and its Arabidopsis orthologue AtAcd1 have been suggested to encode pheide a oxygenase (PaO), a key enzyme catalyzing chlorophyll breakdown. To further elucidate the molecular mechanism that regulates chlorophyll catabolism during soybean leaf senescence, a soybean Lls1 homolog was cloned and designated as GmLls1 (GenBank Accession No. DQ154009). Database searches using the deduced protein sequence revealed that it was highly homologous to Lls1 genes or Lls1 orthologues in Arabidopsis, maize, cowpea and tomato. Structural analysis of the predicted GmLLS1 protein revealed typical Rieske [2Fe-2S] and mononuclear iron-binding domains as well as the C-terminus CxxC motif that were conserved in and featured PaO homologues. RT-PCR results showed that the transcription of GmLls1 was up-regulated in all the three tested senescence systems: the natural leaf senescence process, the dark-induced primary leaf senescence and senescing cotyledons. We have previously described the involvement of an LRR receptor-like kinase (RLK), RLPK2 in regulation of soybean leaf senescence. Here we report that the expression of GmLls1 gene was dramatically down-regulated by the RNAi-mediated suppression of rlpk2 and, as expected, greatly up regulated by the CaMV 35S promoter derived overexpression of this RLK gene. These results suggested that the expression of GmLls1 was controlled by the RLPK2-mediated senescence signaling pathway. The observation that the detached rlpk2-RNAi transgenic leaves exhibited light-dependent necrotic lesions, which featured the lls1 mutation, further supported this hypothesis.

RNAi-mediated knocking-down ofrlpk2 gene retarded soybean leaf senescence

Leaf senescence that occurs in the last stage of leaf development is a genetically programmed process. It is very significant to elucidate the molecular mechanisms that control the initiation and progression of leaf senescence and the way the senescence signal is transduced. In a previous study on artificially induced soybean leaf senescence, we cloned a novel gene designatedrlpk2 (Genbank Accession No. AY687391) that encodes a leucine-rich repeat (LRR) receptor like protein kinase. The expression level ofrlpk2 gene was shown to be strongly up-regulated during both the natural leaf senescence process in this report and the artificially induced primary-leaf-senescence process in our previous work. The RNA interference (RNAi)-mediated knocking-down ofrlpk2 dramatically retarded both the natural and nutrient deficiency-induced leaf senescence in transgenic soybean. The transgenic leaves showed more cell-aggregated surface structure and higher content of chlorophyll.

RNAi-mediated knocking-down of rlpk2 gene retarded soybean leaf senescence

Leaf senescence that occurs in the last stage of leaf development is a genetically programmed process. It is very significant to elucidate the molecular mechanisms that control the initiation and progression of leaf senescence and the way the senescence signal is transduced. In a previous study on artificially induced soybean leaf senescence, we cloned a novel gene designated rlpk2 (Genbank Accession No. AY687391) that encodes a leucine-rich repeat (LRR) receptor like protein kinase. The expression level of rlpk2 gene was shown to be strongly up-regulated during both the natural leaf senescence process in this report and the artificially induced primary-leaf-senescence process in our previous work. The RNA interference (RNAi)-mediated knocking-down of rlpk2 dramatically retarded both the natural and nutrient deficiency-induced leaf senescence in transgenic soybean. The transgenic leaves showed more cell-aggregated surface structure and higher content of chlorophyll.

Water Stress during Seed Filling and Leaf Senescence in Soybean

AbstractWater stress during seed filling could reduce soybean [Glycine max (L.) Merr.] yield by accelerating leaf senescence and shortening the seed filling period. Greenhouse experiments were conducted to (i) evaluate the effect of water stress during seed fill on leaf senescence and (ii) determine the potential role of redistributed C and N in moderating the effect of water stress during seed fill. Soybean plants (‘McCall’, Exp. 1; ‘Elgin 87’, Exp. 2) were grown in soil‐filled pots. At the beginning of Growth Stage R6, two levels of water stress were imposed until maturity: daily waterings of 60% (moderate stress) and 30% (severe stress) of the water required to bring well‐watered pots to field capacity. Moisture stress reduced yield by reducing seed size (8–20%) in Exp. 1 and seed number (18%) and seed size (14–32%) in Exp. 2. Moisture stress accelerated leaf senescence, as shown by more rapid declines in leaf chlorophyll and N, and shortened the seed filling period in both experiments. In Exp. 2, a 50% depodding treatment was applied at the beginning of Growth Stage R6. Depodding delayed senescence and physiological maturity. Moisture stress accelerated leaf senescence and reduced seed size on the depodded plants, but the effect was less than on the nondepodded plants. Our results demonstrate that moisture stress during seed filling reduces yield by accelerating senescence and shortening the seed filling period. Increasing the source‐sink ratio may reduce the effect of moisture stress on seed size.

Carbon and Nitrogen Supply during Seed Filling and Leaf Senescence in Soybean

Leaf senescence and N redistribution during seed filling may limit soybean [Glycine max (L.) Merr.] yield by restricting the seed filling period. Experiments were conducted in the field and greenhouse to evaluate the relationship between seed N demand and leaf senescence. Plants (cv. McCall) were grown under shade cloth (which reduced insolation by 63%) from R1 to R5 (beginning seed fill), then the shade was removed from some plants with the objective of increasing photosynthesis, seed dry matter, and N accumulation. Experiments included plants grown in soil with no nodules (NO−3 as primary source), plants grown in soil with many nodules (N from NO−3 and N2 fixation), and in a hydroponics system (NO−3 as primary N source) where N was removed from the media at R5. Increasing photosynthesis at R5 increased seed dry matter accumulation and the response was larger when N was available. Seed N accumulation also increased if N was available. Increasing photosynthesis had no effect on seed N accumulation when N was not available to the plant. Temporal changes in chlorophyll and N of an upper leaf were measured to characterize leaf senescence. Increasing photosynthesis did not accelerate leaf senescence in any of the treatments. If N was available, N uptake increased when photosynthesis increased. If N was not available, seed N concentration decreased. The data suggest that leaf senescence does not occur because of seed N demand, but may be regulated by processes in the leaf.

Photosynthesis, Rubisco Activity and Amount, and Their Regulation by Transcription in Senescing Soybean Leaves.

Senescence is a phase of leaf ontogeny marked by declining photosynthetic activity that, in soybean (Glycine max [L.] Merr.), is paralleled by a decline in chloroplast function. Soybean leaves have different patterns of decline in photosynthetic capacity and chloroplast function associated with nodal position and sink activity. The objective of this work was to determine whether leaves from nodes 3 and 6 of soybean, which show these different patterns, are similarly regulated with respect to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity and content and also to ascertain the degree of regulation of Rubisco content by transcription. Leaves from nodes 3 and 6 of field-grown soybean plants were sampled periodically from the time of their unfolding until near death. In situ CO2-exchange rate (CER) increased to a maximal level in both leaves and then declined slowly. For node 3 leaves the decline was progressive, but for node 6 leaves the decline was arrested at about 75% of maximum CER for a period of about 20 d, coincident with the onset of rapid seed growth, before a short period of very rapid decline immediately preceding leaf death. Rubisco activities and Rubisco content were directly correlated with CER in the leaves exhibiting the two different patterns. Rubisco activation ratio was similar for the two leaves and did not change throughout development. The primary regulator of photosynthesis at the physiological level, thus, was the amount of Rubisco protein. Decreases in Rubisco holoenzyme during senescence of both leaves were accompanied by coordinate decreases in the levels of mRNAs for the small and large subunits of Rubisco, suggesting that the decrease in Rubisco enzyme amounts during soybean leaf senescence is due to slower transcription rates and that levels of these mRNAs are coordinately controlled during senescence as they are during chloroplast development. However, plastid DNA template availability and posttranscriptional controls may also influence Rubisco content during senescence of these leaves. We conclude that soybean leaf photosynthesis likely unfolds according to a single developmental program but that modifications can be superimposed upon this program to maximize photosynthetic rates.

Cytokinin biochemistry in relation to leaf senescence. III. The senescence-retarding activity and metabolism of 9-substituted 6-benzylaminopurines in soybean leaves

A group of 14 9-substituted derivatives of 6-benzylaminopurine (BA), including the alanine conjugate, oxygen heterocyclic and alkyl derivatives, and compounds with a modified 9-ribose moiety, were assessed for their ability to retard soybean leaf senescence. The 9-alanine conjugate was very weakly active, and only two compounds, 9-(2-tetrahydropyranyl)-BA (9THP-BA) and 9-(2-tetrahydrofuranyl)-BA (9THF-BA), proved to be considerably more effective than BA. The metabolism of these three BA derivatives was determined to rationalize their differing activity. The alanine conjugate of BA was largely unmetabolized in leaf discs, but 9THP-BA and 9THF-BA released free BA and were also debenzylated to 9THP-adenine and 9THF-adenine, respectively. The three products of metabolism were identified by mass spectrometry. The enhanced activity of 9THP-BA and 9THF-BA, relative to that of BA, is attributed to their greater stability and their ability to gradually release free BA. This released BA was less susceptible to inactivation by alanine conjugate formation than was exogenous BA. The novel BA analogue 7-benzylaminooxazolo[5,4-d]pyrimidine, in which the 9-NH is replaced by oxygen, was inactive at 100 μM.