Cytokinin biochemistry in relation to leaf senescence. VIII. Translocation, metabolism and biosynthesis of cytokinins in relation to sequential leaf senescence of tobacco

Cytokinin bases (zeatin and dihydrozeatin) and ribosides (zeatin riboside and dihydrozeatin riboside) were identified as major cytokinins in tobacco xylem sap by radioimmunoassay. When ³H-labelled zeatin riboside or dihydrozeatin riboside were supplied to tobacco plants via the xylem, leaves of differing maturity did not differ appreciably in level of radioactivity or in metabolism of the cytokinin. The major metabolites of zeatin riboside in leaves were adenine, adenosine and adenine nucleotides, whereas that of dihydrozeatin riboside was dihydrozeatin 7-glucoside. Incorporation of [¹⁴C]adenine into zeatin was evident in upper green leaves. indicating that young leaves have the capacity to synthesize cytokinins in situ. In contrast, fully expanded green leaves and senescing tobacco leaves exhibited little or no incorporation of [¹⁴C]adenine into cytokinins. This difference in cytokinin biosynthetic capacity may contribute to the differing cytokinin levels in leaves of different matirity, and may participate in control of sequential leaf senescence in tobacco.     

Movement to bark and metabolism of xylem cytokinins in stems of Lupinus angustifolius

Following uptake of [(3)H]zeatin riboside and [(3)H]dihydrozeatin riboside by girdled lupin (Lupinus angustifolius L.) stems via the transpiration stream, rapid lateral movement of the radioactivity from xylem to bark was observed. Short-term studies with intact stems, and other studies with excised stem tissues, revealed that the ribosides and/or the corresponding nucleotides were the cytokinin forms which actually moved into the bark tissues. Relative to cytokinin metabolism in xylem plus pith, metabolism in bark was both more rapid and more complex. Riboside cleavage and formation of the O-acetylzeatin and O-acetyldihydrozeatin ribosides and nucleotides were almost completely confined to bark tissues. Exogenous (3)H-labelled O-acetylzeatin riboside was converted to zeatin riboside in bark tissue, but the presence of the acetyl group suppressed degradation to adenine metabolites. The sequestration and modification of xylem cytokinins by stem tissues probably contributes significantly to the cytokinin status of the shoot. New cytokinins identified by mass spectrometry in lupin were: O-acetyldihydrozeatin 9-riboside, a metabolite of exogenous dihydrozeatin riboside in stem bark; O-methylzeatin nucleotide and O-methyldihydrozeatin 9-riboside, metabolites of endogenous cytokinins in stem bark; O-methylzeatin nucleotide and O-methylzeatin 9-riboside, metabolites of exogenous zeatin riboside in excised pod walls.     

Cytokinins in the vascular saps of Ricinus communis

Roots are recognised as the major sites of cytokinin synthesis and shoots receive a continuous supply of cytokinins from the roots. Although reports are available on the xylem mobility of putative free bases and their ribosides, relatively few studies on the phloem mobility of cytokinins have been reported. The origin of phloem-mobile cytokinins is uncertain but there is evidence which implicates a recirculation from the root source. This study is the first report in which zeatin and zeatin riboside from the root pressure exudate and phloem sap of Ricinus have been identified by full-scan GC-MS and quantified by GC-MS selective-ion-monitoring. In this study, the concentration of cytokinins in root pressure exudate was similar, but lower, and in the phloem sap higher than that reported previously. The concentration of cytokinins quantified in the phloem sap confirms their transport in the sieve tubes. The relatively high concentration of zeatin riboside detected in the root pressure exudate and of zeatin detected in the phloem sap indicate a possible vascular recirculation of these hormones.     

Cytokinin biochemistry in relation to leaf senescence. VII. Endogenous cytokinin levels and exogenous applications of cytokinins in relation to sequential leaf senescence of tobacco

The cytokinin complex in tobacco leaves of various maturities was characterized by radioimmunoassay and mass spectrometry. Zeatin was the major base, whereas zeatin riboside was identified as the main riboside. in leaves of all maturities studied. Relative to upper younger leaves, the basal yellow leaves had reduced levels of both cytokinin bases and ribosides. Exogenous applications of dihydrozeatin and zeatin to detached tobacco leaves in amounts sufficient to delay senescence, elevated cytokinin base and riboside levels 2–5 fold. Presenescent and senescent leaves of intact plants showed quantitatively similar changes in cytokinin content. which therefore appear to be of significance in control of senescence. When supplied exogenously, the principal cytokinin bases found to occur in tobacco leaves (zeatin and dihydrozeatin) were markedly more effective than auxins and gibberellic acid in retarding senescence. Localised application of cytokinins to leaf blades of detopped plants was much less effective than application to intact plants. The cytokinin induced senescence retardation in tobacco leaves was independent of effects on directed metabolite transport. Evidence that endogenous levels of active cytokinins in intact tobacco leaves are involved in control of sequential leaf senescence is discussed.     

Cytokinins of dryZea mays seed: Quantification by radioimmunoassay and gas chromatography-mass spectrometry

The endogenous cytokinins present in dryZea mays seed were determined using both radioimmunoassay and gas chromatography—mass spectrometry. Similar values for bases and ribosides were obtained by the two methods. The cytokinins present in embryo and endosperm were estimated separately using radioimmunoassay; similar levels of cytokinins were found in these two tissues. The major cytokinins detected on a whole-seed basis were dihydrozeatin riboside, O-glucosyldihydrozeatin riboside, zeatin 9-glucoside, zeatin, and the nucleotides of zeatin, dihydrozeatin, and isopentenyladenine. Cytokinin levels in the mature dry seed were considerably lower than cytokinin levels published in the literature for immature seed. Unexpected activity in the radioimmunoassays was detected in the wash from the DEAE cellulose column chromatography step. The compound(s) responsible for this activity did not have the solvent partitioning characteristics of a cytokinin base or riboside. They eluted as a single fraction following high-performance liquid chromatography on a Zorbax C8 column; this fraction showed no activity in theAmaranthus bioassay for cytokinins, but inhibited the activity of authentic zeatin riboside present at an optimal concentration.     

Ontogenetic variation of four cytokinins in soybean root pressure exudate

Cytokinins exported from the root may be involved in the correlative control of plant development. To test this hypothesis in soybean ((Glycine max [L.] Merr. cv. McCall, cv Chippewa 64, and cv Hodgson 78), cytokinins were intercepted en route from the root to the shoot by collecting root pressure exudate from detopped roots. The quantities of four cytokinins in the exudate were studied throughout the development of plants grown in the field and in controlled environment chambers. Zeatin, zeatin riboside, and their dihydro derivatives, dihydrozeatin and dihydrozeatin riboside, were isolated and quantitated using high-performance liquid chromatography.

Cytokinin fluxes (pmoles per plant per hour) were independent of exudate flux (grams per plant per hour). All fluxes are averages for a 6- or 8-h collection period. The ribosides accounted for the majority of the observed cytokinin transport. The fluxes of zeatin riboside and dihydrozeatin riboside increased from low levels during vegetative growth to maxima during late flowering or early pod formation. Before the seeds began rapid dry matter accumulation, zeatin riboside and dihydrozeatin riboside fluxes decreased and remained at low levels through maturation. The fluxes of zeatin and dihydrozeatin were low throughout development.

No correlation was found between cytokinin fluxes and nodule dry weight or specific nodule activity (acetylene reduction).

The timing of distinct peaks in zeatin riboside and dihydrozeatin riboside fluxes during flowering or pod formation suggests that cytokinins exported from the root may function in the regulation of reproductive growth in soybean.

     

Radioimmunoassay for quantifying the cytokininscis-zeatin andcis-zeatin riboside and its application to xylem sap samples

An antiserum against the cytokinincis-zeatin riboside was raised in rabbits and characterized for use in radioimmunoassays. Cross-reactivity studies demonstrated the specificity of the selected antiserum forcis-zeatin riboside andcis-zeatin in preference to a range of cytokinins and other purines. HPLC systems were developed that separatedcis-zeatin andcis-zeatin riboside from zeatin/dihydrozeatin and zeatin riboside/dihydrozeatin riboside, respectively. These systems enabled the separation of these compounds in xylem sap samples of wheat and oats and their quantification using radioimmunoassay. A TLC system for the separation ofcis-zeatin andcis-zeatin riboside from zeatin/dihydrozeatin and zeatin riboside/dihydrozeatin riboside, respectively, is also described.     

Loading and translocation of various cytokinins in phloem and xylem of the seedlings of Ricinus communis L.

The phloem sap of Ricinus seedlings was analyzed for cytokinins and the concentration was compared with that in cotyledons and xylem sap. The dominant cytokinin in the phloem sap was isopentenyladenine (70 nM) when the endosperm was attached to the cotyledons; zeatin, dihydrozeatin and cytokinin-ribosides were present at relatively low concentrations (1–2 nM). Removal of the endosperm and incubation of the cotyledons in buffer led to a sharp decrease in the level of isopentenyladenine in the phloem sap, down to the value for zeatin, namely 1–2 nM. Similar low cytokinin concentrations were found in the xylem sap, too, whereas in the cotyledons the cytokinin content was at least 10-fold higher. Incubation of the cotyledons with various cytokinins (isopentenyladenine, zeatin and their ribosides) led to an increase of each of the applied cytokinins in the phloem sap, including also the metabolically closely related cytokinins. Zeatin was especially well loaded. It is concluded that the phloem translocates most free bases and ribosides of the various cytokinin species, if they are offered to the phloem. The data also show that the cytokinin levels in the phloem, which may be far higher than in the xylem, are subject to strong fluctuations depending on the physiological situation.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137). The experimental assistance by P. Geigenberger and the help in cytokinin analysis by Dr. A. Fußeder, Dr. B. Wagner, W. Peters (all Bayreuth) and by Prof. E. Weiler (Bochum) is gratefully acknowledged. Also the constructive discussions with Profs. E. Weiler (Bochum) and E. Beck (Bayreuth) are much appreciated.     

Cytokinins in Tobacco and Wheat Chloroplasts. Occurrence and Changes Due to Light/Dark Treatment

Although cytokinins (CKs) affect a number of processes connected with chloroplasts, it has never been rigorously proven that chloroplasts contain CKs. We isolated intact chloroplasts from tobacco (Nicotiana tabacum L. cv SR1) and wheat (Triticum aestivum L. cv Ritmo) leaves and determined their CKs by liquid chromatography/tandem mass spectroscopy. Chloroplasts from both species contained a whole spectrum of CKs, including free bases (zeatin and isopentenyladenine), ribosides (zeatin riboside, and isopentenyladenosine), ribotides (isopentenyladenosine-5'-monophosphate, zeatin riboside-5'-monophosphate, and dihydrozeatin riboside-5'-monophosphate), and N-glucosides (zeatin-N(9)-glucoside, dihydrozeatin-N(9)-glucoside, zeatin-N(7)-glucoside, and isopentenyladenine-N-glucosides). In chloroplasts there was a moderately higher relative amount of bases, ribosides, and ribotides than in leaves, and a significantly increased level of N(9)-glucosides of zeatin and dihydrozeatin. Tobacco and wheat chloroplasts were prepared from leaves at the end of either a dark or light period. After a dark period, chloroplasts accumulated more CKs than after a light period. The differences were moderate for free bases and ribosides, but highly significant for glucosides. Tobacco chloroplasts from dark-treated leaves contained zeatin riboside-O-glucoside and dihydrozeatin riboside-O-glucoside, as well as a relatively high CK oxidase activity. These data show that chloroplasts contain a whole spectrum of CKs and the enzymatic activity necessary for their metabolism.     

Identification of Cytokinins from Xylem Exudate of Phaseolus vulgaris L

The principal biologically active cytokinins in xylem exudate of young Phaseolus vulgaris L. plants were identified by bioassay, high-performance liquid chromatography, enzymic degradation and combined gas chromatography-mass spectrometry (selected ion monitoring) a zeatin riboside, zeatin nucleotide, dihydrozeatin riboside, dihydrozeatin nucleotide, O-glucosyl zeatin, O-glycosyl dihydrozeatin, O-glucosyl dihydrozeatin riboside, and O-glucosyl dihydrozeatin nucleotide. Trace amounts of O-glucosyl zeatin riboside and O-glucosyl zeatin nucleotide were also detected.     

Cytokinins in the Phloem Sap of White Lupin (Lupinus albus L.)

Cytokinin-like activity in samples of xylem and phloem sap collected from field-grown plants of white lupin (Lupinus albus L.) over a period of 9 to 24 weeks after sowing was measured using the soybean hypocotyl callus bioassay following paper chromatographic separation. The phloem sap was collected from shallow incisions made at the base of the stem, the base of the inflorescence (e.g. stem top), the petioles, and the base and tip of the fruit. Xylem sap was collected as root exudate from the stump of plants severed a few centimeters above ground level. Concentration of cytokinin-like substances was highest in phloem sap collected from the base of the inflorescence and showed an increase over the entire sampling period (from week 10 [61 nanogram zeatin equivalents] to week 24 [407 nanogram zeatin equivalents]). Concentrations in the xylem sap and in the other phloem saps were generally lower. Relatively high concentrations of cytokinin-like substances in petiole phloem sap (70 to 130 nanogram zeatin equivalents per milliliter) coincided in time with high concentrations in sap from the base of the inflorescence (see above). Concentrations in sap (phloem or xylem) from the base of the stem were very much lower. This finding is consistent with movement of cytokinins from leaves into the developing inflorescence and fruit, rather than direct input to the fruit from xylem sap. However, an earlier movement of cytokinins from roots into leaves via the xylem cannot be ruled out. Sap collected at an 18-week harvest was additionally separated by sequential C₁₈ reversed-phase high performance liquid chromatography → NH₂ normal phase high performance liquid chromatography, bioassayed, and then analyzed by electron impact gas chromatography-mass spectrometry. Identification of zeatin riboside and dihydrozeatin as two of the major cytokinins in combined sap samples was accomplished by gas chromatography-mass spectrometry-selected ion monitoring.     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

Influence of Different Cytokinins on the Germination of Lettuce (Lactuca sativa) and Celery (Apium graveolens) Seeds

The naturally occurring cytokinins, zeatin, zeatin riboside and dihydrozeatin did not promote the germination of celery (Apium graveolens L.) seeds and 6-Δ²-isopentenyladenine (2iPA) and its riboside were only moderately active. Of the synthetic cytokinins, kinetin, kinetin riboside, and the disubstituted urea, N-phenyl-N′-pyridyl urea (NC5392) were moderately active, and 6-benzyl-aminopurine (BA) and its derivatives BA riboside and 6-benzyl-amino-9(tetrahydropyran-2yl)purine (SD8339) were the most active cytokinins tested. 6-(o-hydroxybenzyl)aminopurine (hyd-BA) and its naturally occurring riboside inhibited germination under normally inductive conditions. All the cytokinins examined were more active in promoting germination of lettuce (Lactuca sativa L.) than celery seeds. BA, BA riboside and SD8339 were again the most active cytokinins. In contrast to the results with celery, zeatin and zeatin riboside were highly active. The other cytokinins also showed high activity with the exception of dihydrozeatin, hyd-BA and hyd-BA riboside which were less active. Cytokinin ribosides were less active than the corresponding free bases during the early period of the lettuce seed incubation but total germination after 90 h was similar.     

Correlation of xylem sap cytokinin levels with monocarpic senescence in soybean

Cytokinins (CKs) coming from the roots via the xylem are known to delay leaf senescence, and their decline may be important in the senescence of soybean (Glycine max) plants during pod development (monocarpic senescence). Therefore, using radioimmunoassay of highly purified CKs, we quantified the zeatin (Z), zeatin riboside (ZR), the dihydro derivatives (DZ, DZR), the O-glucosides, and DZ nucleotide in xylem sap collected from root stocks under pressure at various stages of pod development. Z, ZR, DZ, and DZR dropped sharply during early pod development to levels below those expected to retard senescence. Pod removal at full extension, which delayed leaf senescence, caused an increase in xylem sap CKs (particularly ZR and DZR), while depodding at late podfill, which did not delay senescence, likewise did not increase the CK levels greatly. The levels of the O-glucosides and the DZ nucleotide were relatively low, and they showed less change with senescence or depodding. The differences in the responses of individual CKs to senescence and depodding suggest differences in their metabolism. Judging from their activity, concentrations and response to depodding, DZR and ZR may be the most important senescence retardants in soybean xylem sap. These data also suggest that the pods can depress CK production by the roots at an early stage and this decrease in CK production is required for monocarpic senescence in soybean.     

Methyl jasmonate down-regulates endogenous cytokinin levels in cotyledons of Cucurbita pepo (zucchini) seedlings

Endogenous cytokinins (CK) were measured in Cucurbita pepo L. (zucchini) cotyledons after spraying 7-day-old seedlings with methyl jasmonate (MeJA) to examine whether the senescence-promoting action of MeJA is associated with changes in metabolic regulation of CK levels. MeJA promoted senescence as estimated by the loss of chlorophyll and injury to chloroplast ultrastructure. This was already detected one day after treatment. The contents of physiologically active CKs determined by high-performance liquid chromatography-mass spectrometry (CK bases: trans -zeatin, dihydrozeatin and isopentenyladenine and their ribosides), especially trans -zeatin and its riboside declined considerably. CK nucleotides and physiologically inactive CK 7- and 9-glucosides were also markedly decreased whereas the content of storage CK O -glucosides as well as cis -isomers of zeatin declined only slightly. Chlorophyll content and the normal chloroplast structure recovered 5 days after the MeJa treatment. The process of photosynthetic apparatus recovery correlated with an increased concentration of physiologically active CKs, especially Z. In contrast, the content of CK nucleotides and storage CK O -glucosides decreased further. We suggest that the ability of MeJA to promote some aspects of senescence in intact zucchini cotyledons is at least partially due to down-regulation of endogenous CK levels and their interconversion between active and inactive forms.     

Cloning an ipt gene from Agrobacterium tumefaciens: characterisation of cytokinins in derivative transgenic plant tissue

The isopentenyl transferase gene was isolated from Agrobacterium tumefaciens AcH5 using polymerase chain reaction and transformed into Petunia and Kalanchoë using both A. tumefaciens and A. rhizogenes transformation systems. Morphological evidence and elevated endogenous cytokinin levels indicated that the PCR product was an active gene. Accurate quantification of the cytokinins was obtained by radioimmunoassay, following purification and separation of the free bases and ribosides by HPLC. Of the six cytokinins quantified, zeatin riboside and its stabilised dihydro-derivative, dihydrozeatin riboside, showed the greatest increases in the transformed Petunia tissue (up to 600-fold). The importance of measuring changes in individual cytokinins is discussed.     

Cytokinin Biochemistry in Relation to Leaf Senescence: IV. Cytokinin Metabolism in Soybean Explants

When [³H]dihydrozeatin riboside and [³H]zeatin riboside were supplied to soybean (Glycine max L.) explants (comprising one leaf, associated pods, and subtending stem) via the xylem at mid to late podfill, 0.1% of the supplied ³H was extracted from the seeds. The distribution of ³H in the explants was similar to that bound previously following uptake of [³H]zeatin riboside at earlier stages of pod development. Metabolites formed in the explants from ³H-labeled zeatin, zeatin riboside, and dihydrozeatin riboside were identified and related to the endogenous cytokinins shown to be present. When zeatin riboside and zeatin were supplied for 1 hour, zeatin nucleotide was the principal metabolite formed and this appeared to be the precursor of the other metabolites detected subsequently. Explants supplied with zeatin riboside or dihydrozeatin riboside for 1 hour, and then transferred to water for 20 to 24 hours, yielded leaf blades in which the main metabolites were O-glucosyldihydrozeatin, adenosine, and adenine. The metabolism of zeatin riboside in blades of explants at pre-podfill, early podfill, and mid to late podfill did not differ appreciably. The results are discussed in relation to leaf senescence and seed development.     

Effect of the nitrogen-containing salt on the content of cytokinins in detached wheat leaves

The dynamics of the cytokinin content in detached leaves of wheat (Triticum durum, cv. Bezenchukskaya 139) seedlings moistened with ammonium nitrate or water (control) was studied by immunoenzyme analysis. Leaf treatment with water was accompanied by a transient accumulation of cytokinins, maybe due to their release from their O-glucosylated forms. An increase in the contents of zeatin and its riboside after their initial decrease in detached leaves treated with ammonium nitrate could not occur due to their release from stored forms (nucleotides or O-glucosides) because the contents of zeatin and its riboside increased simultaneously with the content of stored cytokinins. The accumulation of isopentenyladenosine and zeatin nucleotide, which occurred simultaneously with an increase in the content of zeatin and zeatin riboside, permits a supposition that cytokinins can be synthesized in detached wheat leaves treated with ammonium nitrate.     

Stem girdling influences concentrations of endogenous cytokinins and abscisic acid in relation to leaf senescence in cotton

Many studies have shown that root–shoot imbalance influences vegetative growth and development of cotton (Gossypium hirsutum L.), but few have examined changes in leaf senescence and endogenous hormones due to stem girdling. The objective of this study was to determine the correlation between some endogenous phytohormones, particularly cytokinins and abscisic acid (ABA), and leaf senescence following stem girdling. Field-grown cotton plants were girdled on the main stem 5 days after squaring (DAS), while the non-girdled plants served as control. Plant biomass, seed cotton yield, main-stem leaf photosynthetic (Pn) rate, chlorophyll (Chl) and malondialdehyde (MDA) concentrations, as well as levels of cytokinins and ABA in main-stem leaves and xylem sap were determined after girdling or at harvest. Main-stem girdling decreased the dry root weight and root/shoot ratio from 5 to 70 days after girdling (DAG) and reduced seed cotton yield at harvest. Main-stem leaf Pn and Chl concentration in girdled plants were significantly lower than in control plants. Much higher levels of MDA were observed in main-stem leaves from 5 to 70 DAG, suggesting that stem girdling accelerated leaf senescence. Girdled plants contained less trans-zeatin and its riboside (t-Z + t-ZR), dihydrozeatin and its riboside (DHZ + DHZR), and isopentenyladenine and its riboside (iP + iPA), but more ABA than control plants in both main-stem leaves and xylem sap. These results suggested that main-stem girdling accelerated leaf senescence due to reduced levels of cytokinin and/or increased ABA. Cytokinin and ABA are involved in leaf senescence following main-stem girdling.     

Cytokinin Activity in Lupinus albus

The cytokinin content of the root exudate and leaves of fruiting white lupin plants (Lupinus albus L.) was investigated at 2 weekly intervals after anthesis of the lowest flower on the primary inflorescence. Up to 8 weeks after anthesis the level of cytokinins in the root exudate increased. However, at 10 weeks after anthesis insufficient sap was produced for analysis. Cytokinins co-eluting with zeatin and zeatin riboside were detected in the root exudate after fractionation on Sephadex LH-20. The cytokinin levels in the mature leaves steadily increased up to 8 weeks after anthesis and thereafter remained relatively constant. Three peaks of activity, co-eluting with zeatin, zeatin riboside and the glucoside cytokinins were recorded in the leaf extracts. The level of glucoside cytokinins in the leaves was high at 8 and 10 weeks after anthesis. Paper chromatography of extracts of fruits collected at 2 weeks after anthesis indicated that as fruit development proceeded there was a build up of cytokinin in this region of the plant. It is suggested that, in the white lupin, the cytokinins translocated to the shoot are accumulated in the leaves and in the fruits and that it is only later when there is a considerable decrease in sap (10 weeks after anthesis) production that a decreasing supply of cytokinins leads to shoot senescence.