Stipule morphologies of the sinuate leaf (sil) mutants
Husbands, A., Emirzade, T. and
Botany and Plant Sci., Univ. of California
Riverside, CA 92521, USA
Form and development of the
pinnately compound leaf of pea are
genetically controlled by a number
of known genes (6). Some genes
appear to affect only blade
development and others to affect
only stipule development. The best
known blade mutations are afila (af),
which transforms the proximal
leaflets into branched tendrils; and,
tendrilless (tl), which transforms the
distal tendrils into leaflets (4). A
mutation that affects both blade and
stipule, known as sinuate leaf (sil),
was originally recognized in an af
line. af/af sil/sil plants possess
"adventitious tendrils arising from a
cleft in the distal tip of stipules"
(Fig. la; 5). Marx (5) originally
Fig. 1. sil stipule phenotypes. a. stipule from lower node showing
adventitious tendrils forming from cleft at stipule tip as originally described
by Marx (5); b. stipules at flowering node showing no phenotype. Bars =
2.5 cm. B — leaf blade: F — flower buds: P — Petiole: S — stipule.
described the phenotypes of sil
mutants in Af/Af, Af/af and af/af backgrounds. He reported that Af/- sil plants had sinuate or undulated
leaflet and stipule margins but that only af/af sil plants had stipule tip modifications. He concluded that there
were clear gene interactions between Af and Sil, but that anatomical or histological studies were necessary to
develop a better understanding of them. In an attempt to address Marx's challenge, we recreated these three
lines and started making observations of stipule form changes during shoot ontogeny in preparation for
selecting material for anatomical preparations. Here we correct Marx's observations on stipular morphologies
present on Af/Af sil/sil and Af/af sil/sil plants and provide a hypothesis to explain the interactions between Af
Materials and Methods
Two sil lines, W6 15137 (A Af b er f fs I pal sil) and W6 15110 (a af i sil) were obtained from the Marx
collection at the USDA Western Regional Plant Introduction Station, Pullman, Washington and were the
parental lines in this study. W6 15110 was used as the female and W6 15137 was used as the male parent.
The F1 plants were grown and allowed to self. F3 seedlings from individual F2 plants were phenotyped to
identity the genotype of the parental line. Seed was separated into three genotypic categories: Af/Af sil/sil,
af/af sil/sil, and Af/- sil/sil. For observations and photography, plants were grown in a standard greenhouse in
the fall (short days) when the stipular phenotypes were most dramatic. F4 seeds of each genotype were
planted during the first week of October and developed under natural lighting conditions. Greenhouse
temperatures were approximately 20° in the day and 15° at night.
Distinct morphological features were characteristic of stipule tips on plants of each of the genotypes
generated in this study. There was a progressive change in expression from node to node, up the plant. On
adult flowering plants, the stipules at lower, or juvenile, nodes displayed nearly normal phenotypes, which
increased in severity up to the last two preflowering nodes, which typically displayed the most extreme
phenotypes. The leaves subtending flowers had a normal stipule phenotype (Fig. lb). For each of the
genotypes listed below we describe
phenotypes typical of lower, middle
and upper (preflowerjng) nodes.
Af/Af sil/sil plants - At lower and
middle nodes, many stipules had a
greener, diamond-shaped zone at
their tips, which increased in size at
successive nodes (Figs. 2a,b). At
upper nodes, stipules had clefts or
were deeply bisected due to the
presence of adventitious leaflet-like
structures (Fig. 2c). These central
structures were deeper green than the
Af/af sil/sil plants - At lower to
middle nodes, these stipules also had
greener distal tips, or terminal clefts,
often with leaflet-like structures
present within them (Figs. 2d,e).
The central leaf-like structure was
darker green than the remainder of
the stipule. At upper nodes, stipule
clefts possessed terminal, adventitious
wildtype leaves with leaflets and
tendrils (Fig. 2f). Leaflet and tendril
pairing on these adventitious leaves
was irregular. The complexity (i.e.
the number of leaflets and tendrils
along the rachis) of these
adventitious "leaves" increased at
successive nodes. Cleft depth also
increased with adventitious leaf
complexity. The most complex leaf-
like structures had their "petioles"
positioned at the node on the shoot.
This bisected the stipules into two
unequal parts. Therefore it appeared
that the stipule had been converted
into a wildtype leaf with its own
af/af sil/sil plants - Some juvenile
stipules were adaxially curled from
Fig. 2. sil phenotypes across various Af backgrounds, (α-c). Af sil
genotype, a. stipule tip matches the greener color associated with leaflets
(lower nodes); b. stipule tip colored and slightly elongated (middle nodes);
c. stipule tip is deeply bisected with adventitious leaflet-like structure, (d-
f). Af/af sil genotype, d. adventitious leaflet-like structure developing in
apical stipule cleft (lower nodes); e. stipule partially converted into
trifoliate structure (middle nodes); f. stipule converted to wildtype leaf,
complete with own stipules (upper nodes), (g-i). af sil genotype, g.
juvenile stipules showing contortion at tips (lower nodes); h. stipule
converted into fully developed af leaf (upper nodes); i. expanding adult
puckered, contorted "knots" on the
midrib about 1/2 to 2/3s of the way up
leaf showing conversion occurring at early stages of leaf development. Bars = 2.5 cm. B - leaf blade; L - leaflet; P - petiole; S - stipule.
(arrows, Fig. 2g). Some stipules were
adaxially bent (*, Fig. 2g). These stipules typically also had greener tips. At middle nodes, the stipules
possessed clefts with "adventitious tendrils" or "adventitious af leaves" emanating from the base of the cleft
(Fig. la). Like on Af/af sil/sil plants, the cleft became deeper and the af "leaf" became larger and more
complex at upper nodes until in the most extreme cases the stipules appeared to be converted into af leaves,
bisecting the stipule into two unequal parts (Fig. 2h). This stipule to leaf conversion occurred very early in
leaf development as illustrated by an expanding adult leaf (Fig. 2i). The adventitious, stipular leaves were an
accurate replica of true af leaves since they possessed branched tendrils at proximal positions and simple
tendrils at distal ones.
More leaf form mutants are known for pea than probably any other species of plant. In addition, these
mutants span a phenomenal range of phenotypes. The range of phenotypic expression and extent of gene
interactions associated with the sil mutants have not been appreciated in the past. The sil phenotype shows
some similarity to another stipule mutant, cochleata. In light of evidence that A/ and Tl show gradients of
expression in pea leaves (3,4,7) and that auxin plays important roles in pea leaf morphogenesis (2), we
propose a new interpretation of Sil action and its interaction with A/.
Clearly Marx missed the fact that Af/Af and Af/af plants display characteristic sil stipule phenotypes. His
omission may be because expression differs in different pea backgrounds, but more importantly we have noted
that expression is much more evident in short day growing conditions than long day. A range of expression
occurs both on a single plant (i.e. heteroblastic expression) and across the genotypes. Expression is greater for
the Af/af and af/af genotypes compared to Af/Af, so the presence of at least one recessive af allele is necessary
for strong expression. It is possible that sil is directly influenced by daylength, which has not been tested.
However, we hypothesize that the increased expression in short days is related to the fact that under these
conditions more vegetative leaves are produced per plant and that the last formed vegetative leaves show the
greatest expression. Leaves associated with flowering do not show sil expression in any of these genotypes.
The range of sil expression across the genotypes can be categorized. The patterns of expression that occur
in at least two genotypes are: (i) greener patches at the stipule tips, (ii) presence of "adventitious leaves" at
the distal tip, and (iii) conversion of the stipule into a leaf. Two other observations made previously by Marx
(5) support our interpretation that the novel structures produced in the latter two expression categories are
leaves in the true sense. First, Marx (5) illustrated the phenotype of af sil tl plants and showed that the
"adventitious leaves" are morphologically similar to the leaves on af tl plants, in that they possessed branched
pinnae with miniature leaflets. These observations demonstrate that two leaf form mutations, af and tl, have
the same effect in these novel stipule-derived structures on sil plants as they do in true leaves. Further, Marx
(5) showed that in combination with two different wax mutants, wb and wlo, the amount of wax deposited on
the tips vs. the base of sil stipules differed and that the amount of wax on the tips was more similar to that on
leaflets for both wax mutants. In addition, we interpret the greener stipule tips on sil mutants to possess leaf
blade identity, because leaflets have less wax on their surfaces than stipules and the observed color difference
appears to be due to wax deposition. So we interpret the three categories of sil expression to represent a
transition from stipule to leaf conversion. The stipules in the first two categories are mosaics of leaf blade and
stipule identities and the final stage represents a complete conversion to a leaf. SEM and anatomical studies
are in progress to test this hypothesis.
The sil mutant shares some phenotypic similarities and many differences with the cochleata (coch) mutant
of pea. Both these mutants have compound stipules resembling true leaves at middle and upper, preflowering
nodes. The compound stipules on coch leaves are conversions to leaf blades only, because they do not possess
"stipules" of their own (8) as the sil mutants do. No interaction with Af is necessary for this conversion on
coch plants, and no mosaic intermediates have been described, coch mutants have abnormal flowers with
reduced fertility (8), whereas sil mutants have normal, fertile flowers.
Auxin has long been known to be produced in developing leaf primordia and to be transported basipetally
through developing shoots. Cells respond to auxin and depending on their position and status, undergo cell
division, elongation or differentiation. The tips and margins of developing leaves are thought to be sources of
auxin (1). Since stipule tips and margins are most affected by sil, we predict that the combination of af and sil
causes excessive auxin accumulation, due to increased synthesis and/or defective transport. In this high auxin
environment, the developing cells of the stipule perceive that they are leaf primordia instead of stipule
primordia and follow an altered developmental path. We hope to test this hypothesis experimentally.
The sil mutants of pea are characterized by sinuate leaflet and stipule margins and, in certain genetic
backgrounds, conversion of the stipule into a stipule/leaf blade mosaic or into a leaf. A distinct stipule tip
phenotype for sil mutants is present in all Af/af backgrounds, and it shows a heteroblastic progression of
severity for each genotype. Conversion can range from slight differences in wax deposition to complete
conversion of the stipule into a compound leaf with its own stipules. The extent of this conversion depends
on various factors including daylength and nodal position. More dramatic conversions are evident when
plants are grown in short days. Due to the fact that the sil mutation primarily affects margin and tip growth,
the Sil gene may be involved in auxin perception or transport.
Acknowledgement: This work was supported by a grant from USDA/CSREES 2001-35304-10958 to D.A.D.
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